Common and rare genetic variations associated with common variable immunodeficiency (CVID) and methods of use thereof for the treatment and diagnosis of the same

ABSTRACT

Compositions and methods useful for the diagnosis and treatment of common variable immunodeficiency are disclosed.

This application is a continuation in part application of PCT/US2011/051385 filed Sep. 13, 2011 which in turn claims priority to U.S. Provisional Application 61/382,231 filed Sep. 13, 2010, which is incorporated herein by reference as though set forth in full.

This invention relates to the fields of genetics and the diagnosis of common variable immunodeficiency (CVID). More specifically, the invention provides compositions and methods useful for the diagnosis and treatment of CVID.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited through the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Common variable immunodeficiency (CVID) disorders are manifested by insufficient quantity and quality of immunoglobulin leading to susceptibility to bacterial infections¹. CVID is considered to be a primary immunodeficiency disease (PIDD) as it is believed to result from intrinsic deficits affecting immunological functions. CVID is heterogeneous in presentation, presenting either early or later in life and associated with a group of known comorbidities². Efforts to subcategorize CVID in order to predict outcomes and comorbid conditions both clinically and by immunologic phenotypes are ongoing³. BAFFR⁴, TACI^(5,6,7), and certain HLA haplotypes^(8,9) have been identified as potential gene candidates for susceptibility to CVID. ICOS^(10,11), CD81¹², CD19^(13,14), CD20¹⁵, harbor disease causing mutations that presently explain only a small percentage of cases. The heterogeneous presentations of patients with CVID make management of these conditions difficult and searches for novel genetic predictors of disease causation or susceptibility have been of limited success.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods are provided for the diagnosis and treatment of CVID. An exemplary method entails detecting the presence of at least one CVID associated CNV in a target polynucleotide wherein if said CNV(s) is/are present, said patient has an increased risk for developing CVID.

In one aspect of the present invention, a method for detecting a propensity for developing common variable immunodeficiency disorder (CVID) in a patient in need thereof is provided. An exemplary method entails detecting the presence of at least one SNP containing nucleic acid in a target polynucleotide, said SNP being informative of a the presence of a CVID associated copy number variation (CNV), wherein if said SNP is present, said patient has an increased risk for developing CVID, wherein said SNP containing nucleic acid is provided in Tables 1 to 5. In one embodiment, at least 1, 2, 3, 4, 5, 10, 15, 20 or all of the SNPs provided herein are detected.

In another embodiment of the invention a method for identifying agents which alter immune cell function or signaling is provided. Such a method comprises providing cells expressing at least one nucleic acid comprising the CVID associated CNVs of the invention, (step a); providing cells which express the cognate wild type sequences which lack the CNV (step b); contacting the cells from each sample with a test agent and analyzing whether said agent alters immune signaling or function of cells of step a) relative to those of step b), thereby identifying agents which alter immune cell signaling or function. Methods of treating CVID patients via administration of test agents identified using the methods described herein are also encompassed by the present invention. The invention also provides at least one isolated CVID related SNP-containing nucleic acid selected from the group listed in Tables 1 to 5. In one embodiment, a multiplex SNP panel containing all of the informative SNPs from the tables provided herein is disclosed. Such SNP containing nucleic acids which indicate the presence of CVID associated CNV(s) may optionally be contained in a suitable expression vector for expression in immune cells. Alternatively, they may be immobilized on a solid support. In yet another alternative embodiment, the panel may be provided in silico.

According to yet another aspect of the present invention, there is provided a method of treating CVID in a patient determined to have at least one prescribed single nucleotide polymorphism indicative of the presence of a CVID-associated copy number variation, as described hereinbelow, by administering to the patient a therapeutically effective amount of an agent useful for modulating immune function. This method provides a test and treat paradigm, whereby a patient's genetic profile is used to personalize treatment with therapeutics targeted towards specific gastrointestinal defects found in individuals exhibiting CVID. Such a test and treat model may benefit up to 50% of patients with CVID with greater efficacy and fewer side effects than non-personalized treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. SNP based case:control significance is shown in negative log base 10 genome wide for Discovery (FIG. 1A) and Replication (FIG. 1B and FIG. 1C) inclusive CVID cohorts. Subsequently, CVID cases with specific disease subphenotypes were compared to CVID cases without the subphenotype. The single SNP tests are shown as single points. Multiple neighboring SNPs of similar significance boost confidence in the association as shown in the strong peak on 6p22.1-p21.32 of the Discovery case:control cohort. Conversely the median significance P value is kept low by minimizing population stratification which minimizes the genomic inflation factor.

FIGS. 2A-2B. Frequency of CNV in cases (FIG. 2A) and controls (FIG. 2B). CNVs are called on a single sample basis with deletions and duplications in specific genomic regions based on the genotype and intensity signal of contiguous SNPs. These single sample CNV profiles are plotted as a SNP-based statistic to allow for SNP-based association testing. Lastly, neighboring SNPs with similar significance define a CNVR. Light grey indicates case deletion; Dark grey indicates case duplication; Black indicates control deletion; Medium grey indicates control duplication.

FIG. 3. Duplication of ORC4L Found Exclusive to 15 CVID Cases. Vertical lines indicate the SNP probe coverage. Rectangles delineate regions of copy number variation in individual cases. Three exons of ORC4L are shown duplicated in 15 cases and 0 controls. It is likely that the duplications could extend to a larger region of ORC4L based on coverage.

FIGS. 4A-4K. CNV Validation with Illumina Intensity Data Review and Independent Array Technology, Affymetrix 2.7M

FIG. 5. The core diagnosis of CVID has many clinical progressions with varying frequency. The height and size of subphenotypes signify the frequency of the specific progression. Given significant SNP genotype associations for each CVID subphenotype progression vs. CVID patients without the progression, prediction can be made which may improve clinical outcome.

FIG. 6. Schematic Representation of Observed Data with Classification Divided by a Hyperplane. Binary SVM selects a hyperplane (bold line) that maximizes the width of the ‘gap’ (margin) between the two classes. The hyperplane is specified by ‘boundary’ training instances, called support vectors shown with circles. New cases are classified based on which side of the hyperplane they are mapped.

FIG. 7. SVM Model for CVID Case vs. individual not affected by CVID.

FIG. 8. SVM Model for CVID case with Bronchiectasis versus CVID case without Bronchiectasis.

FIG. 9. SVM Model for CVID case with OSAI (organ specific autoimmunity) versus CVID case without OSAI.

FIG. 10. Pedigree of a family with autosomal dominant common variable immunodeficiency

FIG. 11. SVM Algorithm Hyperplane.

FIG. 12. S535N mutation in IRF2BP2.

FIG. 13. RT-PCR or IRF2BP2.

DETAILED DESCRIPTION OF THE INVENTION

Common variable immunodeficiency disorders (CVID) are a group of uncommon heterogeneous immune defects characterized by hypogammaglobulinemia, failure of antibody production, susceptibility to bacterial infections and an array of serious comorbidities. To address the underlying immunopathogeneses of CVID, we conducted the first genome-wide association study of patients with CVID. 363 patients were genotyped with 610,000 SNPs. Due to the relative rarity of this group of disorders, the study cohort was recruited at four sites. Patients were randomly divided into a discovery cohort of 179 cases in comparison with 1,917 disease-free controls and a replication cohort of 109 cases in comparison with 1,114 controls, controlled for population stratification. Our analyses detected strong association with the MHC region and uncovered a novel association with a cluster of ADAM genes that was replicated in the independent case cohort. Analysis of the same cases for copy number variation (CNV) revealed 16 disease-associated deletions and duplications, as well as numerous unique rare intraexonic deletions and duplications suggesting multiple novel genetic etiologies for individual CVID cases. Analysis of CVID comorbidities identified significantly associated SNP genotypes with the major CVID clinical phenotypes. Taken together, our integrative genome-wide analysis of SNP genotypes and CNVs has uncovered multiple novel susceptibility loci for CVID, both common and rare, consistent with the highly heterogeneous nature of CVID. These results may allow for improved diagnosis of CVID, prediction of the CVID clinical phenotypes, and mechanistic insights into immune diseases based upon these unique genetic variations.

I. Definitions

For purposes of the present invention, “a” or “an” entity refers to one or more of that entity; for example, “a cDNA” refers to one or more cDNA or at least one cDNA. As such, the terms “a” or “an,” “one or more” and “at least one” can be used interchangeably herein. It is also noted that the terms “comprising,” “including,” and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.

The term “genetic alteration” as used herein refers to a change from the wild-type or reference sequence of one or more nucleic acid molecules. Genetic alterations include without limitation, base pair substitutions, additions and deletions of at least one nucleotide from a nucleic acid molecule of known sequence.

A “single nucleotide polymorphism (SNP)” refers to a change in which a single base in the DNA differs from the usual base at that position. These single base changes are called SNPs or “snips.” Millions of SNP's have been cataloged in the human genome. Some SNPs such as that which causes sickle cell are responsible for disease. Other SNPs are normal variations in the genome. Frequently SNPs are used to “mark” particular genes and genetic regions, particularly those containing CNVs for example.

A “copy number variation (CNV)” refers to the number of copies of a particular gene or segment thereof in the genome of an individual. CNVs represent a major genetic component of human phenotypic diversity. Susceptibility to genetic disorders is known to be associated not only with single nucleotide polymorphisms (SNP), but also with structural and other genetic variations, including CNVs. A CNV represents a copy number change involving a DNA fragment that is ˜1 kilobases (kb) or larger. CNVs described herein do not include those variants that arise from the insertion/deletion of transposable elements (e.g., ˜6-kb KpnI repeats) to minimize the complexity of future CNV analyses. The term CNV therefore encompasses previously introduced terms such as large-scale copy number variants (LCVs), copy number polymorphisms (CNPs), and intermediate-sized variants (ISVs), but not retroposon insertions. The terminology “duplication-containing CNV” is also used herein below consistent with the CNV definition provided.

“CVID-associated SNP” or “CVID-associated specific marker” is a SNP or marker which is associated with an increased or decreased risk of developing CVID not found normal patients who do not have this disease. Such markers may include but are not limited to nucleic acids, proteins encoded thereby, or other small molecules. Thus, the phrase “CVID-associated SNP containing nucleic acid” is encompassed by the above description.

The term “solid matrix” as used herein refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter. The material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose.

The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.

The phrase “partial informative CNV” is used herein to refer to a nucleic acid that hybridizes to sequences comprising a duplication on a chromosome however, the partial informative CNV may not be identical to the duplication, rather, the CNV may correspond to only a portion of the duplication, but yet is still informative for the presence of the same.

“Target nucleic acid” as used herein refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined wild-type region contains at least one known nucleotide variation which may or may not be associated with CVID. The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer. With regard to nucleic acids used in the invention, the term “isolated nucleic acid” is sometimes employed. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived. For example, the “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote. An “isolated nucleic acid molecule” may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted into a vector is also sometimes referred to herein as a recombinant nucleic acid molecule.

With respect to RNA molecules, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form.

By the use of the term “enriched” in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that “enriched” does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.

It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term “purified” in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level, this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10⁻⁶-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Thus the term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest.

The term “complementary” describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a “complement” of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine, cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.

With respect to single stranded nucleic acids, particularly oligonucleotides, the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. For example, specific hybridization can refer to a sequence which hybridizes to any CVID specific marker gene or nucleic acid, but does not hybridize to other nucleotides. Also polynucleotide which “specifically hybridizes” may hybridize only to a single specific marker, such as an CVID-specific marker shown in the Tables contained herein. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.

For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989): T _(m)=81.5° C.+16.6 Log[Na+]+0.41(% G+C)−0.63 (% formamide)−600/# bp in duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5° C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated T_(m) of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20° C. below the T_(m) of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. A very high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

The term “oligonucleotide,” as used herein is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide. Oligonucleotides, which include probes and primers, can be any length from 3 nucleotides to the full length of the nucleic acid molecule, and explicitly include every possible number of contiguous nucleic acids from 3 through the full length of the polynucleotide. Preferably, oligonucleotides are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length.

The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein.

The term “vector” relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.

Many techniques are available to those skilled in the art to facilitate transformation, transfection, or transduction of the expression construct into a prokaryotic or eukaryotic organism. The terms “transformation”, “transfection”, and “transduction” refer to methods of inserting a nucleic acid and/or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like.

The term “promoter element” describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. In one embodiment, the promoter element of the present invention precedes the 5′ end of the CVID specific marker nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.

Those skilled in the art will recognize that a nucleic acid vector can contain nucleic acid elements other than the promoter element and the CVID specific marker nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification.

A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, plastid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.

An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.

As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.

The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.

The term “selectable marker gene” refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell.

The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.

The terms “recombinant organism” or “transgenic organism” refer to organisms which have a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The term “organism” relates to any living being comprised of a least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, the phrase “a recombinant organism” encompasses a recombinant cell, as well as eukaryotic and prokaryotic organism.

The term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.

A “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules. Examples of specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.

“Sample” or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, preferably a CVID specific marker molecule, such as a marker described hereinbelow. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, gastric lavage, saliva, tears, pleural fluid and the like.

The terms “agent” and “compound” are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide/DNA complexes, and any nucleic acid based molecule which exhibits the capacity to modulate the activity of the CNV or SNP-containing nucleic acids described herein or their encoded proteins. Agents and compounds may also be referred to as “test agents” or “test compounds” which are evaluated for potential biological activity by inclusion in screening assays described hereinbelow.

The term “modulate” as used herein refers to increasing/promoting or decreasing/inhibiting a particular cellular, biological or signaling function associated with the normal activities of the genetic alteration containing molecules described herein or the proteins encoded thereby. For example, the term modulate refers to the ability of a test compound or test agent to interfere with signaling or activity of a gene or protein of the present invention. Alternatively, the term may refer to augmentation of the activity of such a protein.

II. Methods of Using CVID-Associated CNVs and/or SNPs for Diagnosing a Propensity for the Development of CVID

The present invention provides methods of diagnosing CVID in a patient or methods for identifying a patient having an increased risk of developing CVID. Diagnosis, as used herein, includes not only the initial identification of CVID-associated with the genetic alterations described herein in a patient but confirmatory testing, or screening in patients who have previously been identified as having or likely to have CVID. The methods include the steps of providing a biological sample from the patient, measuring the amount of particular sets (e.g., those with the highest statistical significance), 1, 2, 3, 4, 5, 10, 10, 20 or all of the CVID associated markers (Tables herein) present in the biological sample, preferably a tissue and/or blood plasma sample, and determining if the patient has a greater likelihood of having or developing CVID based on the amount and/or type of CVID marker expression level determined relative to those expression levels identified in patient cohorts of known outcome. A patient has a greater likelihood of having CVID when the sample has a CNV marker expression profile associated with patients previously diagnosed with CVID. The compositions and methods of the invention are useful for the prognosis and diagnosis and management of CVID.

In another aspect, the patient sample may have been previously genotyped and thus the genetic expression profile in the sample may be available to the clinician. Accordingly, the method may entail storing reference CVID associated marker sequence information in a database, i.e., those CNVs statistically associated with a more favorable or less favorable prognosis as described in the tables herein, and performance of comparative genetic analysis on the computer, thereby identifying those patients having increased risk of CVID.

CVID-related CNV or SNP-containing nucleic acids, including but not limited to those listed below may be used for a variety of purposes in accordance with the present invention. CVID-associated CNV or SNP-containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of CVID specific markers. Methods in which CVID specific marker nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).

Further, assays for detecting CVID-associated CNVs or SNPs may be conducted on any type of biological sample, including but not limited to body fluids (including blood, urine, serum, gastric lavage, cerebral spinal fluid), any type of cell (such as brain cells, white blood cells, mononuclear cells, fetal cells in maternal circulation) or body tissue.

Clearly, CVID-associated CNV or SNP-containing nucleic acids, vectors expressing the same, CVID CNV or SNP-containing marker proteins and anti-CVID specific marker antibodies of the invention can be used to detect CVID associated CNVs or SNPs in body tissue, cells, or fluid, and alter CVID CNV or SNP-containing marker protein expression for purposes of assessing the genetic and protein interactions involved in the development of CVID.

In most embodiments for screening for CVID-associated CNVs or SNPs, the CVID-associated CNV or SNP-containing nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are important in the art.

Alternatively, new detection technologies can overcome this limitation and enable analysis of small samples containing as little as 1 μg of total RNA. Using Resonance Light Scattering (RLS) technology, as opposed to traditional fluorescence techniques, multiple reads can detect low quantities of mRNAs using biotin labeled hybridized targets and anti-biotin antibodies. Another alternative to PCR amplification involves planar wave guide technology (PWG) to increase signal-to-noise ratios and reduce background interference. Both techniques are commercially available from Qiagen Inc. (USA).

Any of the aforementioned techniques may be used to detect or quantify CVID-associated CNV or SNP marker expression and accordingly, diagnose an increased risk for developing the same.

III. Kits and Articles of Manufacture

Any of the aforementioned products can be incorporated into a kit which may contain a CVID-associated CNV or SNP specific marker polynucleotide or one or more such markers immobilized on a solid support or Gene Chip, provided in silico in a database, one or more oligonucleotides, a polypeptide, a peptide, an antibody, a label, marker, reporter, a pharmaceutically acceptable carrier, reagents suitable for performance of PCR with positive and negative control nucleic acids, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, an assay substrate, or any combination thereof.

IV. Methods of Using CVID-Associated CNVs and/or SNPs for the Development of Therapeutic Agents

Since the CNVs and SNPs identified herein have been associated with the etiology of CVID, methods for identifying agents that modulate the activity of the genes and their encoded products containing such CNVs and/or SNPs should result in the generation of efficacious therapeutic agents for the treatment of this disorder.

Several regions of the human genome provide suitable targets for the rational design of therapeutic agents. Small nucleic acid molecules or peptide molecules corresponding to these regions may be used to advantage in the design of therapeutic agents that effectively modulate the activity of the encoded proteins.

Molecular modeling should facilitate the identification of specific organic molecules with capacity to bind to the active site of the proteins encoded by the CNV or SNP-containing nucleic acids based on conformation or key amino acid residues required for function. A combinatorial chemistry approach will be used to identify molecules with greatest activity and then iterations of these molecules will be developed for further cycles of screening. Molecules available for testing in this screening assay, include without limitation, Apilimod Mesylate (STA-5326), an oral small-molecule compound that selectively inhibits the production of the IL-12 family of proteins Inhibition of IL-production may improve the gastrointestinal manifestations of CVID. Apilimod mesylate selectively inhibits this pathway and reduces over-production of IL-12 and IL-23. Other agents that may have therapeutic utility for treating CVID symptoms include the ACVR2A antagonist Sotaercept and the ACVR antibody PF3446962 as ACVR2A exhibits a significant CNV duplication associated with CVID. PEG-interleukin-2 and B-lymphocyte stimulators may also be screened for therapeutic utility in the treatment of CVID. The polypeptides or fragments employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may determine, for example, formation of complexes between the polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between the polypeptide or fragment and a known substrate is interfered with by the agent being tested.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity for the encoded polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds, such as those described above, are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the target polypeptide and washed. Bound polypeptide is then detected by methods well known in the art.

A further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional or altered CVID associated gene. These host cell lines or cells are defective at the polypeptide level. The host cell lines or cells are grown in the presence of drug compound. Altered immune signaling or function of the host cells is measured to determine if the compound is capable of regulating this function in the defective cells. Host cells contemplated for use in the present invention include but are not limited to bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells. However, mammalian cells, particularly immune cells are preferred. The CVID-associated CNV or SNP encoding DNA molecules may be introduced singly into such host cells or in combination to assess the phenotype of cells conferred by such expression. Methods for introducing DNA molecules are also well known to those of ordinary skill in the art. Such methods are set forth in Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y. 1995, the disclosure of which is incorporated by reference herein.

A wide variety of expression vectors are available that can be modified to express the novel DNA sequences of this invention. The specific vectors exemplified herein are merely illustrative, and are not intended to limit the scope of the invention. Expression methods are described by Sambrook et al. Molecular Cloning: A Laboratory Manual or Current Protocols in Molecular Biology 16.3-17.44 (1989). Expression methods in Saccharomyces are also described in Current Protocols in Molecular Biology (1989).

Suitable vectors for use in practicing the invention include prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif. 92037), pET vectors (Novogen Inc., 565 Science Dr., Madison, Wis. 53711) and the pGEX vectors (Pharmacia LKB Biotechnology Inc., Piscataway, N.J. 08854). Examples of eukaryotic vectors useful in practicing the present invention include the vectors pRc/CMV, pRc/RSV, and pREP (Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif. 92121); pcDNA3.1/V5&His (Invitrogen); baculovirus vectors such as pVL1392, pVL1393, or pAC360 (Invitrogen); and yeast vectors such as YRP17, YIPS, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.); Picchia vectors such as pHIL-D1 (Phillips Petroleum Co., Bartlesville, Okla. 74004); retroviral vectors such as PLNCX and pLPCX (Clontech); and adenoviral and adeno-associated viral vectors.

Promoters for use in expression vectors of this invention include promoters that are operable in prokaryotic or eukaryotic cells. Promoters that are operable in prokaryotic cells include lactose (lac) control elements, bacteriophage lambda (pL) control elements, arabinose control elements, tryptophan (trp) control elements, bacteriophage T7 control elements, and hybrids thereof. Promoters that are operable in eukaryotic cells include Epstein Barr virus promoters, adenovirus promoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMV) promoters, baculovirus promoters such as AcMNPV polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promoter, as well as neuronal-specific platelet-derived growth factor promoter (PDGF), the Thy-1 promoter, the hamster and mouse Prion promoter (MoPrP), and the Glial fibrillar acidic protein (GFAP) for the expression of transgenes in glial cells.

In addition, a vector of this invention may contain any one of a number of various markers facilitating the selection of a transformed host cell. Such markers include genes associated with temperature sensitivity, drug resistance, or enzymes associated with phenotypic characteristics of the host organisms.

Host cells expressing the CVID-associated CNVs and/or SNPs of the present invention or functional fragments thereof provide a system in which to screen potential compounds or agents for the ability to modulate the development of CVID. Thus, in one embodiment, the nucleic acid molecules of the invention may be used to create recombinant cell lines for use in assays to identify agents which modulate aspects of cellular metabolism associated with CVID and aberrant glutaminergic function. Also provided herein are methods to screen for compounds capable of modulating the function of proteins encoded by CNV and SNP-containing nucleic acids.

Another approach entails the use of phage display libraries engineered to express fragment of the polypeptides encoded by the CNV or SNP-containing nucleic acids on the phage surface. Such libraries are then contacted with a combinatorial chemical library under conditions wherein binding affinity between the expressed peptide and the components of the chemical library may be detected. U.S. Pat. Nos. 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology 9:19-21. In one approach, discussed above, the three-dimensional structure of a protein of interest or, for example, of the protein-substrate complex, is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., (1990) Science 249:527-533). In addition, peptides may be analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390-411). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based.

One can bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original molecule. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of polypeptide activity. By virtue of the availability of CNV or SNP-containing nucleic acid sequences described herein, sufficient amounts of the encoded polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.

In another embodiment, the availability of CVID-associated CNV or SNP-containing nucleic acids enables the production of strains of laboratory mice carrying the CVID-associated SNPs or CNVs of the invention. Transgenic mice expressing the CVID-associated CNV or SNP of the invention provide a model system in which to examine the role of the protein encoded by the CNV or SNP-containing nucleic acid in the development and progression towards CVID. Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and 3. the incorporation of genetically manipulated embryonic stem cells into an early embryo. Production of the transgenic mice described above will facilitate the molecular elucidation of the role that a target protein plays in various cellular metabolic processes, including: aberrant antibody production and function, altered immunoreceptor ligand signaling and aberrant responses to bacterial and viral infections. Such mice provide an in vivo screening tool to study putative therapeutic drugs in a whole animal model and are encompassed by the present invention.

The term “animal” is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A “transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term “transgenic animal” is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term “germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.

The alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. Such altered or foreign genetic information would encompass the introduction of CVID-associated CNV or SNP-containing nucleotide sequences.

The DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.

A preferred type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction. The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.

One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated CVID-associated CNV or SNP genes as insertional cassettes to selectively inactivate a wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice. The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).

Techniques are available to inactivate or alter any genetic region to a mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles. However, in comparison with homologous extrachromosomal recombination, which occurs at a frequency approaching 100%, homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10⁻⁶ and 10⁻³. Nonhomologous plasmid-chromosome interactions are more frequent occurring at levels 10⁵-fold to 10² fold greater than comparable homologous insertion.

To overcome this low proportion of targeted recombination in murine ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening of individual clones. Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly. One of the most powerful approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes for which no direct selection of the alteration exists. The PNS method is more efficient for targeting genes which are not expressed at high levels because the marker gene has its own promoter. Non-homologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with effective herpes drugs such as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou-racil, (FIAU). By this counter selection, the number of homologous recombinants in the surviving transformants can be increased. Utilizing CVID-associated CNV or SNP-containing nucleic acid as a targeted insertional cassette provides means to detect a successful insertion as visualized, for example, by acquisition of immunoreactivity to an antibody immunologically specific for the polypeptide encoded by CVID-associated CNV or SNP nucleic acid and, therefore, facilitates screening/selection of ES cells with the desired genotype.

As used herein, a knock-in animal is one in which the endogenous murine gene, for example, has been replaced with human CVID-associated CNV or informative fragment thereof or SNP-containing gene of the invention. Such knock-in animals provide an ideal model system for studying the development of CVID.

As used herein, the expression of a CVID-associated CNV or SNP-containing nucleic acid, partial informative CNV fragment thereof, or an CVID-associated fusion protein in which the CNV or SNP is encoded can be targeted in a “tissue specific manner” or “cell type specific manner” using a vector in which nucleic acid sequences encoding all or a portion of an CVID-associated CNV or SNP are operably linked to regulatory sequences (e.g., promoters and/or enhancers) that direct expression of the encoded protein in a particular tissue or cell type. Such regulatory elements may be used to advantage for both in vitro and in vivo applications. Promoters for directing tissue specific proteins are well known in the art and described herein.

Methods of use for the transgenic mice of the invention are also provided herein. Transgenic mice into which a nucleic acid containing the CVID-associated CNV or SNP or its encoded protein have been introduced are useful, for example, to develop screening methods to screen therapeutic agents to identify those capable of modulating the development of CVID.

V. Pharmaceutical and Peptide Therapies

The elucidation of the role played by the CVID associated CNVs and SNPs described herein in immunoreceptor ligand signaling and function facilitates the development of pharmaceutical compositions useful for treatment and diagnosis of CVID. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.

The following materials and methods are provided to facilitate the practice of the present invention.

Patients

The diagnosis of CVID was established in concordance with existing diagnostic criteria^(16,38). All patients were enrolled in institutionally approved research protocols to enable genetic analysis and collection of clinical data. Subsets of the patients reported here have been previously included in published studies^(2,22,39).

Illumina Infinium Assay for SNP Genotyping and CNV Discovery

We performed high-throughput, genome-wide SNP genotyping, using the InfiniumII HumanHap550 BeadChip technology (Illumina San Diego, Calif.), at the Center for Applied Genomics at CHOP. The genotype data content together with the intensity data provided by the genotyping array provides high confidence for CNV calls. Importantly, the simultaneous analysis of intensity data and genotype data in the same experimental setting establishes a highly accurate definition for normal diploid states and any deviation from the norm. To call CNVs, we used the PennCNV algorithm¹², which combines multiple sources of information, including Log R Ratio (LRR) and B Allele Frequency (BAF) at each SNP marker, along with SNP spacing and population frequency of the B allele to generate CNV calls. Rare recurrent CNVs were the focus of our study.

CNV Quality Control

We calculated Quality Control (QC) measures on our HumanHap550 GWAS data based on statistical distributions to exclude poor quality DNA samples and false positive CNVs. The first threshold is the percentage of attempted SNPs which were successfully genotyped. Only samples with call rate >98% were included. The genome wide intensity signal must have as little noise as possible. Only samples with the standard deviation (SD) of normalized intensity (LRR) <0.35 were included. All samples must have Caucasian ethnicity based on principle components analysis and all other samples were excluded. Furthermore, case and control matching was insured by calculating a genomic inflation factor between groups. Wave artifacts roughly correlating with GC content resulting from hybridization bias of low full length DNA quantity are known to interfere with accurate inference of copy number variations. Only samples where the GC corrected wave factor of LRR −0.02<x<0.02 were accepted. If the count of CNV calls made by PennCNV exceeds 100, it is suggestive of poor DNA quality, and those samples were excluded. Thus, only samples with CNV call count <100 were included. Any duplicate samples (such as monozygotic twins or repeats on the same patient) were identified and as a result one sample was excluded.

Statistical Analysis of CNVs

CNV frequency between cases and controls was evaluated at each SNP using Fisher's exact test. We only considered loci that were nominally significant between cases and controls (p<0.05) for which patients had the same variation, that were observed in multiple cohorts or were not observed in any of the control subjects, and were validated with an independent method. We report statistical local minimums to narrow the association in reference to a region of nominal significance including SNPs residing within 1 Mb of each other. Resulting nominally significant CNVRs were excluded if they met any of the following criteria: i) residing on telomere or centromere proximal cytobands; ii) arising in a “peninsula” of common CNV arising from variation in boundary truncation of CNV calling; iii) genomic regions with extremes in GC content which produces hybridization bias; or iv) samples contributing to multiple CNVRs. Three lines of evidence establish statistical significance: independent replication p<0.05, permutation of observations, and no loci observed with control enriched significance. We used DAVID (Database for Annotation, Visualization, and Integrated Discovery)⁴² to assess the significance of functional annotation clustering of independently associated results into InterPro categories.

The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

Example 1

Genome wide single nucleotide polymorphism (SNP) arrays have enabled high throughput typing of genomic DNA with tagging of the whole genome based on linkage disequilibrium. Given both intensity and genotype content, copy number variations (CNVs) can also be detected using the same GWAS platforms. Both SNPs and CNVs have a significant difference in frequency between cases and controls in a variety of diseases. We genotyped 363 CVID cases and 3,031 healthy controls on the Illumina Infinium HumanHap550K BeadChip. We sought to associate SNPs and CNVs with CVID¹⁶, as well as with the characteristic clinical and immunologic phenotypes of this syndrome, to address disease heterogeneity. We additionally compared the CVID results with those obtained from Inflammatory Bowel Disease (IBD), another disease with an immunologic etiology but without susceptibility to infection, to serve as a “disease control” cohort and define associations unique to CVID subjects. Included in the cohort were 29 subjects with known mutations in TACI including 10 C104R, 8 A181E, 6 V220A and 1 occurrence of each of C104R/S144X, C104R/S194X, A181E/L171R, R72H, and S194X heterozygous.

Our CVID case cohort was composed of 223 patients from Mount Sinai School of Medicine, 76 patients from University of Oxford, 37 patients from The Children's Hospital of Philadelphia, and 27 patients from University of South Florida. The diagnosis in each case was validated against the ESID/PAGID diagnostic criteria¹⁶ and only patients confirmed by these criteria were included.

We first evaluated quality and suitability of the data for a case control study. The primary parameters were sample based call rates and population based stratification. Seven samples had call rates lower than 98% and were excluded. Based on principle components analysis by Eigenstrat¹⁷, the cases were stratified into three clusters: 179 cases were of confirmed European ancestry and another 109 Caucasian cases clustered separately, but very close to the other Caucasian cluster, whereas 30 samples were significant outliers from Caucasian and were omitted from this analysis. We also observed comparable clustering of the 179 cases to the 1,917 Caucasian controls. However, there were relatively few Caucasian controls that clustered well with the second Caucasian case cluster. To perform a more comprehensive search for additional control individuals that clustered with the latter case cohort, we ran 18,000 controls in 19 batches together with our cases through Eigenstrat. This identified over 1,000 control samples clustering similarly to the second Caucasian case cohort. We then reran those identified samples together with cases to yield a robustly stratified cluster with a total of 1,114 controls. This yielded a discovery cohort of 179 cases and 1,917 controls along with a replication cohort of 109 cases and 1,114 controls. We also excluded individuals with observed cryptic relatedness of genotypes as well as three known and two newly discovered 22q11 deletions. For CNV calling, samples with high noise (standard deviation of Log R Ratio >0.35) in intensity signal were additionally removed.

First, a genome wide association analysis (GWAS) was performed using the SNP genotype data. Genotype frequencies were compared between cases versus controls and a chi-square test statistic applied in Plink¹⁸ for SNPs with at least 90% call rate and 1% minor allele frequency. The discovery cohort of 179 cases and 1,917 controls yielded a low genomic inflation factor of 1.02783 (FIG. 1A). The 1,000 most significant SNPs were evaluated for enrichment in genomic regions to boost confidence in significance based on neighboring SNPs also with significance in the top 1,000. We found 87 such regions with multiple significant SNPs composed of 344 SNPs in total, the remainder of which we excluded as spurious signal (Table 1). The most significant association was the major histocompatibility complex (MHC) with 110 neighboring SNPs showing significance in the top 1,000, the most significant SNP rs3117426 having P=8.62×10⁻¹⁰. A total of 62 regions were supported by two SNPs, and 25 regions were supported by 3 or more SNPs, with 47 regions directly impacting genes while 40 did not.

TABLE 1 Genomic Regions with Multiple SNPs in the 1000 most Significant Associations Distance Region From CountSNPs Best Best Genomic Span Gene Distance Exon Top1000 P SNP F_A F_U chr6: 27236785- MHC , Many 0 0 110  8.62E−10 rs3117426 0.3268 0.1907 32521295 chr8: 23746576- ADAM28, ADAM7 , ADAMDEC1, 0 0 4 6.24E−6 rs4872262 0.03911 0.01069 24681608 STC1 chr6: 42776382- CRIP3, CUL7, GNMT, 0 0 4 1.22E−5 rs422717 0.2961 0.1982 43502348 KLC4, KLHDC3, MEA1, MRPL2, PARC , PEAS, PEX6, PPP2R5D , PRPH2, PTCRA, PTK7 , RPL7L1, SLC22A7, SRF, TBCC, TNRC5 , TTBK1, UNQ1934, ZNF318 chr14: 39744450- BX248273 584047 584047 4 1.69E−5 rs6571989 0.1927 0.115 39909619 chr4: 189671181- AK095968 0 18764 2 1.78E−5 rs1606234 0.2961 0.4124 189676119 chr16: 6120908- A2BP1 0 110914 4 1.85E−5 rs12924882 0.1313 0.06915 6137836 chr2: 224812598- FAM124B 110570 110570 4 1.89E−5 rs4264554 0.3184 0.4353 224841089 chr2: 45599872- PRKCE, SRBD1 0 0 7 2.12E−5 rs17322265 0.3408 0.4576 45895169 chr10: 73083830- CDH23, KIAA1812 0 7287 2 2.45E−5 rs7087554 0.5894 0.4729 73084583 chr2: 74996818- AK125960 7650 7650 2 3.25E−5 rs3771781 0.3184 0.4319 75001224 chr20: 55079605- BMP7 95559 95559 2 4.44E−5 rs6127923 0.2318 0.1498 55083403 chr7: 4270929- SDK1 0 0 5 4.70E−5 rs895710 0.2905 0.4003 4287047 chr20: 15025030- C20orf133 0 50997 4 5.15E−5 rs6043091 0.2151 0.1367 15074507 chr8: 8804291- MFHAS1 15750 15750 3 6.80E−5 rs400404 0.3715 0.2726 8808564 chr1: 158072814- CR625159, RP11- 0 0 2 7.31E−5 rs10494349 0.2067 0.1312 158076453 190A12.4, SLAMF8 chr18: 25213638- CDH2 1202449 1202449 2 7.53E−5 rs9950880 0.2821 0.1943 25220939 chr3: 106535466- ALCAM , CBLB , Nbla00127 0 0 5 8.57E−5 rs13062596 0.4358 0.3328 107174255 chr6: 85682448- TBX18 151830 151830 2 9.07E−5 rs9444253 0.05028 0.1187 85684185 chr1: 20369369- FLJ32784, UBXN10 0 0 7 9.09E−5 rs7514144 0.2654 0.1808 20490791 chr10: 27709830- PTCHD3 0 0 7 9.77E−5 rs506659 0.4022 0.3026 27728115 chr12: 123259607- FAM101A 64652 64652 4 1.01E−4 rs7972182 0.3855 0.2874 123275011 chr13: 68136436- BC042673 195237 195237 2 1.09E−4 rs287355 0.2219 0.3213 68138181 chr16: 69996550- CALB2 14714 14714 2 1.09E−4 rs12102284 0.1676 0.1015 70004357 chr11: 25128661- LUZP2 67899 67899 2 1.10E−4 rs11028465 0.3352 0.2426 25130289 chr12: 49729991- DKFZp586A011, LETMD1 0 214 2 1.22E−4 rs4768959 0.1397 0.08033 49731928 chr4: 25702025- LOC389203 161493 161493 5 1.34E−4 rs2048507 0.257 0.1755 25756263 chr7: 13858986- ETV1 31148 31148 4 1.35E−4 rs12532319 0.09218 0.04617 13866233 chr1: 59987116- FLJ10986, RP11-242B9.1 0 2458 2 1.52E−4 rs11207520 0.3994 0.3026 59993733 chr6: 12070732- HIVEP1 47026 47026 2 1.54E−4 rs12193434 0.09777 0.05034 12073684 chr19: 40814281- MGC10433 0 134 2 1.55E−4 rs2285415 0.352 0.4559 40815243 chr6: 139472753- HECA 20371 20371 2 1.56E−4 rs17304375 0.2318 0.1549 139477571 chr12: 80435676- PPFIA2 0 59900 2 1.63E−4 rs2400955 0.4134 0.3159 80444630 chr12: 69717561- TSPAN8 85073 85073 2 1.63E−4 rs6581986 0.2905 0.2053 69720071 chr11: 102069786- MMP27 0 0 2 1.69E−4 rs17099394 0.1229 0.0687 102079454 chr3: 188168001- ST6GAL1 0 3593 2 1.71E−4 rs12495023 0.2765 0.1934 188168026 chr16: 71592439- ATBF1 0 40845 2 1.80E−4 rs8056528 0.3687 0.2754 71594134 chr8: 124729628- C8ORFK36 0 0 4 2.05E−4 rs4871402 0.06425 0.1325 124734756 chr22: 42908147- KIAA1644, PARVG 0 0 8 2.27E−4 rs80303 0.3994 0.5013 43159207 chr20: 44622587- NADC3, SLC13A3 0 0 5 2.49E−4 rs393990 0.09218 0.1664 44638545 chr1: 9459963- SLC25A33 54611 54611 2 2.57E−4 rs10746490 0.1453 0.2292 9467504 chr3: 7364204- GRM7 0 40865 4 2.96E−4 rs12491592 0.1536 0.0939 7369287 chr11: 46472478- ARHGAP1, 0 0 4 3.19E−4 rs2306029 0.3603 0.4593 46855347 CKAP5, CR612190, F2 , LRP4, MEGF7, ZNF408, coagulation factor II chr2: 181999958- AK125001 27433 27433 2 3.22E−4 rs16867404 0.09497 0.05008 182002374 chr7: 121184414- PTPRZ1 106504 106504 2 3.24E−4 rs1196493 0.5251 0.4266 121193891 chr3: 8378739- BC020876 0 129448 2 3.51E−4 rs359030 0.1648 0.1033 8381919 chr6: 25570803- LRRC16 0 0 2 3.61E−4 rs4320355 0.3799 0.2898 25574868 chr4: 131889955- BC041448 793982 793982 2 3.68E−4 rs2125639 0.2905 0.2094 131893245 chr15: 58159939- FOXB1 74505 74505 2 3.71E−4 rs7168491 0.1648 0.1035 58162165 chr6: 153354039- MTRF1L 0 0 2 3.81E−4 rs9322400 0.4469 0.3526 153355207 chr5: 172969660- FAM44B 0 0 4 3.83E−4 rs258873 0.2849 0.3798 172992688 chr6: 55433518- HMGCLL1 0 21202 2 3.86E−4 rs9382494 0.2179 0.3078 55442340 chr9: 15192417- C9orf52 0 0 2 3.87E−4 rs693196 0.3603 0.2723 15195014 chr4: 20207250- SLIT2 0 0 2 4.29E−4 rs573118 0.243 0.169 20210955 chr3: 284363- CHL1 0 19083 2 4.31E−4 rs17273893 0.148 0.09081 285747 chr11: 45198534- PRDM11 0 650 2 4.36E−4 rs12417962 0.06983 0.1351 45198959 chr3: 138891290- SOX14 74205 74205 2 4.37E−4 rs12637203 0.1844 0.12 138892064 chr8: 121001793- DEPDC6 0 0 2 4.41E−4 rs869340 0.324 0.2402 121010709 chr18: 74518781- SALL3 315148 315148 2 4.72E−4 rs2931060 0.1257 0.07381 74526115 chr4: 143965929- INPP4B 0 14945 2 4.81E−4 rs10000770 0.1788 0.1158 143971867 chr3: 115619659- ZBTB20 0 29017 2 5.19E−4 rs2718419 0.2598 0.1844 115623404 chr2: 65518304- FLJ16124 0 784 2 5.20E−4 rs1194849 0.3659 0.4614 65520560 chr5: 68236398- AK128486 55041 55041 2 5.27E−4 rs7718291 0.1313 0.2081 68245085 chr21: 27713547- BC043580 0 19690 2 5.51E−4 rs469709 0.1704 0.1095 27722878 chr3: 5866648- EDEM1 630006 630006 2 5.91E−4 rs2572690 0.1145 0.06602 5866856 chr16: 7470020- A2BP1 0 33910 2 5.93E−4 rs2191388 0.2709 0.1948 7474240 chr6: 88936647- CNR1 4366 4366 2 6.35E−4 rs9344757 0.2179 0.3041 88941540 chr4: 32018838- PCDH7 1265004 1265004 3 6.41E−4 rs2130904 0.2458 0.3343 32029980 chr1: 202125395- SNRPE 18492 18492 2 6.48E−4 rs12145634 0.1369 0.08346 202130221 chr7: 38733082- HVPS41, VPS41 0 0 2 6.67E−4 rs10255854 0.2179 0.3038 38736264 chr11: 120260865- GRIK4 0 5023 2 6.67E−4 rs12577638 0.2011 0.1356 120269428 chr11: 91278373- FAT3 444245 444245 2 6.97E−4 rs10501763 0.03911 0.09207 91283755 chr16: 20151678- GP2 77031 77031 2 7.14E−4 rs9921767 0.0419 0.09572 20152281 chr1: 221871750- CAPN2 84031 84031 3 7.29E−4 rs3856154 0.4134 0.3254 221882793 chr10: 130929688- MGMT 224087 224087 2 7.33E−4 rs538186 0.05307 0.1104 130931369 chr5: 170727300- NPM1 12168 12168 2 7.52E−4 rs7707008 0.1173 0.1894 170734557 chr10: 63071553- C10orf107 12553 12553 2 7.69E−4 rs10994852 0.1061 0.06051 63080172 chr21: 31427707- TIAM1 0 0 2 7.93E−4 rs2833297 0.2095 0.1435 31432285 chr4: 42434168- ATP8A1 80521 80521 2 8.09E−4 rs6812482 0.2709 0.1965 42438752 chr6: 161106705- PLG 12377 12377 2 8.78E−4 rs9295131 0.2151 0.2986 161109328 chr17: 12323188- AX747308, BC122562 68477 68477 2 9.02E−4 rs8069430 0.2584 0.186 12325534 chr17: 50125991- TOM1L1, tom1-like 205624 205624 2 9.20E−4 rs6504930 0.03371 0.08325 50127579 chr16: 77330083- WWOX 0 305933 2 9.21E−4 rs1364290 0.1844 0.1231 77334376 chr11: 5493737- HBG2, UBQLNL 0 0 4 9.28E−4 rs2047456 0.3436 0.2624 5511794 chr3: 70644812- LOC401072 96929 96929 2 9.91E−4 rs17790790 0.5196 0.4293 70648510 chr3: 1292289- CNTN6 0 0 2 1.06E−3 rs6799262 0.3571 0.2733 1302261 chr3: 64977356- BC040632 5173 5173 2 1.15E−3 rs1517927 0.2318 0.1643 64985877 chr5: 14818187- ANKH 0 0 2 1.15E−3 rs17251715 0.3955 0.3113 14825245

We next sought to replicate these SNPs and the direction of the difference in allele frequency, based on our replication cohort of 109 cases and 1114 controls with a genomic inflation factor of 1.04789 (FIG. 1B). Four SNPs displayed significance (P<0.05) and allele frequency in the same direction as the discovery cohort, including rs11207520, rs1194849, rs17790790, and rs4872262. The associated chromosomal region demonstrating replication that impacted characterized genes was 8p21.2, harboring ADAM28, ADAM7, ADAMDEC1, and STC1. When all SNPs contributing to the discovery region were queried in replication rather than just the most significant SNP, 8 (four additional) SNPs displayed significance (P<0.05) and allele frequency in the same direction as discovery. There were three additional regions with directly affected genes: the MHC, UBXN10, and SDK1 (Table 2); others did not overlap genes directly.

TABLE 2 Most Significant Associated Regions Based on Genotype Association Count Dis- Region SNPs tance P SNP F_A F_U Genomic Top From Repli- Repli- Repli- Repli- Span Gene 1000 Exon P SNP A1 F_A F_U cation cation cation cation chr6: 27236785- MHC 110 0  8.62E−10 rs3117426 T 0.3268 0.1907 0.0004 rs2156875 0.5734 0.4475 32521295 chr8: 23746576- ADAM28, 4 0 6.24E−6 rs4872262 A 0.0391 0.0107 0.0314 rs4872262 0.0321 0.0135 24681608 ADAM7 , ADAMDEC1, STC1 chr7: 4270929- SDK1 5 0 4.70E−5 rs895710 T 0.2905 0.4003 0.0235 rs4720301 0.3991 0.4793 4287047 chr1: 20369369- FLJ32784, 7 0 9.09E−5 rs7514144 C 0.2654 0.1808 2.25E−8 rs6426636 0.4679 0.2857 20490791 UBXN10 chr1: 59987116- FGGY 2 2458 1.52E−4 rs11207520 G 0.3994 0.3026 0.0015 rs11207520 0.2569 0.1706 59993733 chr2: 65518304- FLJ16124 2 784 5.20E−4 rs1194849 G 0.3659 0.4614 0.0119 rs1876518 0.3853 0.4744 65520560 Bold underline: Known immunological function genes, A1: allele 1 which is the minor allele, F_A: Frequency of allele 1 in affected individuals, F_U: Frequency of allele 1 in unaffected individuals. See additional detail inTable 11.

We had access to a large IBD cohort that had been genotyped previously¹⁹. Since this is also a heterogeneous immunological condition but without increased infectious susceptibility, it was considered that this group would be appropriate as a disease control group. Analysis of the 2,413 IBD cases and 6,197 controls revealed a number of signals in the IBD cohort that were distinct from both controls and the CVID cohort (including 19 CVID subjects who had been diagnosed with IBD). These included a signal in DEPDC6 (P<0.05) with allele frequency in the same direction as well as the MHC locus. In addition, TIAM1, SLAMF8, GRIK4, 6p21.1, PPFIA2, SLC25A33, A2BP1, and UBXN10 were also observed in the IBD cohort only (P<0.05). Importantly, however, the majority of associations in CVID identified in comparison to controls were not observed in the IBD cohort, suggesting that that they were not a general feature of an immunologic disease, but specific to the CVID phenotype. The non-overlapping loci between CVID and IBD included FLJ16124, ADAM28-ADAM7-ADAMDEC1-STC1, ANKH, FLJ109860-RP11-242B9.1, and WWOX, which are therefore more likely to lead to loss of B cell and antibody function.

We next performed a CNV analysis, where rare (<1%) CNVs were called, using PennCNV²° and SNP-based CNV frequency in cases and controls; these were compared for associations. We analyzed deletion and duplication CNV frequency of 311 cases and 2,766 controls (FIG. 2). To limit bias due to population stratification, the contribution of patients from the discovery Caucasian cluster defined in the genotype GWAS was required to establish confidence. We also monitored the contribution of each sample subgroup from the four different sample collection sites. We found 5 deletions and 11 duplications to be recurrent and significantly enriched in the patients as compared to controls (Table 3). In addition, a total of 15 regions were exclusive to CVID cases. Duplications of 2q23.1 associated with multiple exons of ORC4L were observed to be exclusive to 15 cases (P=8.66×10⁻¹⁶) (FIG. 3). We also observed large CNVs with at least 10 SNPs exclusive to single CVID cases totaling 84 deletions and 98 duplications in CNVs. (Table 4). No significant overrepresentation was observed in the controls in this frequency range. Of clinical relevance, 2 patients were newly identified with deletion in the 22q11 region suggesting an alternative presentation of the well-characterized PIDD associated with this microdeletion. Of 5 deletions and 23 duplications significantly enriched in CVID, 4 deletions and 21 duplications significant in CVID were not significant in IBD. 10 regions of homozygous deletion were observed exclusively in CVID cases (Table 5). To further prioritize resulting significant loci for potential functional impact, we calculated distance of CNVs from exons to highlight loci impacting exons directly, a method applied previously²¹. We examined overall CNV burden in cases compared to controls for significant results, exonic CNVs, and large (100 kb) rare (<1%) CNVs and found deletions to be significantly enriched in cases when considering CNV observations on all loci genome wide (Table 6). All but 5 of the 182 CNVs exclusive to the CVID cohort were intraexonic. To assess the reliability of our CNV detection method, we reviewed the BAF (genotype) and LRR (intensity) values of our Illumina data and experimentally validated all the significant CNVRs using an independent method, the Affymetrix Cytogenetics Whole-Genome 2.7M Array which provides high resolution with 400,103 SNP and 2,387,595 CN probes (FIG. 4).

TABLE 3 Most Significant Associated Regions Based on CNV Association A) Deletions Count Distance From P Cases Controls Com- CNVR Deletion SNPs Exon Deletion Del Del Gene MSSM Oxford USF bined chr11: 85365857- 5 0 0.01 2 0 PICALM , PICALM variant 0 2 0 2 85381622 protein chr20: 57735790- 6 5902 0.026 3 4 PHACTR3 2 1 0 3 57741780 chr22: 17396663- 270 0 0.029 2 1 ARVCF, COMT , TBX1 , 2 0 0 2 18417315 others chr4: 10256682- 4 2915 0.029 2 1 CLNK 0 1 1 2 10264316 chr10: 46003146- 8 0 0.029 2 1 DKFZp566K0524, 2 0 0 2 46042543 PTPN20A, PTPN20B B) Duplications Count Distance From P Cases Controls Com- CNVR Duplication SNPs Exon Dup Dup Dup Gene MSSM Oxford USF bined chr2: 148396730- 6 0 8.66E−16 15 0 ACVR2A , 10 5 0 15 148433180 ORC4L chr15: 35053039- 6 23146 5.72E−13 19 10 MEIS2 17 2 0 19 35063531 chr7: 91789778- 4 0 1.20E−09 12 4 ANKIB1 9 3 0 12 91801963 chr2: 163316298- 3 17100 1.00E−07 7 0 KCNH7 6 1 0 7 163316595 chr7: 87180702- 4 202 7.41E−07 7 1 RPIB9 5 2 0 7 87191931 chr19: 9256584- 4 0 0.001 3 0 ZNF699 2 0 1 3 9277749 chr4: 39190766- 6 0 0.004 3 1 UGDH 1 2 0 3 39201960 chr7: 18903915- 4 23171 0.009 3 2 HDAC9 1 2 0 3 18905725 chr5: 170531269- 4 374 0.01 2 0 DKFZp666P032, 1 1 0 2 170536993 RANBP17 chr7: 34685030- 4 4517 0.01 2 0 AAA1, NPRS1 1 1 0 2 34686172 chr1: 170400944- 4 26274 0.01 2 0 DNM3 0 1 1 2 170403742 Additional detail in Table 12.

TABLE 4 Large Genic CNVs Impacting Single CVID Cases not Observed in Controls Distance Count From CNVR Del Singleton SNPs Exon Gene A) Deletions Impacting Immune Function Genes chr4: 86650109-87684765 209 0 ARHGAP24, BC038746, MAPK10 chr16: 75457204-76988778 78 0 ADAMTS18, AK026469, BC035731, CLEC3A, KIAA1576, MON1B, WWOX chr3: 74573112-74866049 66 0 CNTN3 chr19: 56625454-56761712 64 0 LOC729767, SIGLEC12 , SIGLEC6 , SIGLEC8 chr4: 103671292-104700248 51 0 AK093356, BDH2, CENPE, CENPE variant protein, CR604221, LOC133308, MANBA, NFKB1 , NHEDC1, UBE2D3, UNQ6308, ZCD2 chr14: 43997374-46152755 45 0 AX748292, BC038722, C14orf106, C14orf155, C14orf28, FANCM, FKBP3 , KIAA0423, KLHL28, PRPF39 chr6: 29844580-30045812 44 0 AK097625, BC035647, HLA-A, HLA-A*0226, HLA-G, HLA- G2.2 , LOC554223, LOC642032, NR_001317, NR_001318, NR_002139 chr2: 86695685-87945980 40 0 ANAPC1, BC066991, CD8A , CD8B , DQ576041, LOC285074, MGC4677, PLGLB1, PLGLB2, RGPD1, RGPD2, RMND5A, RNF103 chr6: 22380732-22525808 40 0 PRL chr6: 31465923-33432505 34 0 AF075059, AGER , AGPAT1, AIF1 , AK057104, AL050203, APOM, ATP6V1G2, B3GALT4, BAT1 , BAT2, BAT3, BAT4 , BAT5 , BF , BRD2 , BTNL2 , C2 , C4A , C4A variant protein, C6orf10, C6orf21 , C6orf25 , C6orf26, C6orf27, C6orf31, C6orf47, C6orf48, CFB , CLIC1, COL11A2, CREBL1, CS266662, CSNK2B, CYP21A2, DAXX , DDAH2, DKFZp313H139, DKFZp547I194, DKFZp779M0311 , DOM3Z, EGFL8, EHMT2, FKBPL , G18.1a, G6E, G6e, G7c, G8, GPSM3, HCP5, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA- DPB1, HLA-DPB2, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA- DRB1, HLA-DRB5, HSD17B8, HSPA1A, HSPA1B , HSPA1L, LOC401252, LSM2, LST1, LTA, LTB, LY6G5B , LY6G5C, LY6G6C, LY6G6D, MCCD1, MEGT1, MICA, MICB, MSH5, NCR3, NEU1, NFKBIL1, NG36/G9a, NOTCH4, NR_002742, NR_002745, NR_002812, NR_002971, NR_003140, PBX2, PFDN6, PP199 , PPT2, PRRT1, PSMB8, PSMB9, RAGE , RDBP, RGL2, RING1, RNF5, RP, RPS18, RXRB, SKIV2L, SLC39A7, SLC44A4, STK19, TAP1, TAP2, TAPBP, TNF, TNFa , TNXB, VARS, VPS52, WDR46, ZBTB12, ZBTB22 chr2: 70616413-71523501 30 0 ADD2, AF090102, ANKRD53, ATP6V1B1, CD207 , CLEC4F, FIGLA, KIAA1155, MCEE, MPHOSPH10, N-Acetylglucosamine kinase, NAGK, NP220, OR7E91P, TEX261, TGFA , VAX2, ZNF638 chr8: 95257759-96161427 28 0 AF086017, AX747981, C8orf38, CCNE2, CDH17 , DPY19L4, FSBP, GEM , INTS8, KIAA1429, RAD54B, RBM35A, TP53INP1, p53DINP1 chr1: 184639488-185615061 24 0 C1orf27, PDC, PLA2G4A , PTGS2 chr4: 124486894-125137357 21 0 AL833449, BC053945, SPRY1 chr1: 226522770-226673588 20 0 KIAA1556, KIAA1639, OBSCN , TRIM11, TRIM17 chr21: 14806346-14824849 17 0 HACS1, SAMSN1 chr7: 84542383-84633646 16 0 SEMA3D chr4: 162635366-163113553 15 0 FSTL5 chr19: 48834611-49626859 15 0 AK090553, AK098175, AK131520, AX748287, BC041923, BC045755, CADM4 , CR593740, DKFZp564H1322, FLJ12886, HZF19, HZF6, IRGC , KCNN4, LYPD5, PLAUR ZNF155, ZNF221, ZNF222, ZNF223, ZNF224, ZNF225, ZNF226, ZNF227, ZNF228, ZNF229, ZNF230, ZNF233, ZNF234, ZNF235, ZNF284, ZNF285A, ZNF404, ZNF45 chr4: 90603695-91299621 14 0 AK123890, CR605611, KIAA1680, MGC48628, MMRN1, SNCA chr6: 138020053-138532047 14 0 AK124173, PERP, TNFAIP3 chr7: 30225447-30798217 14 0 AK096056, AK096687, AK097240, AL137445, BC016976, BC041636, BX648714, C7orf24, CR595731, CRH2R, CRHR2 , FLJ22374, GARS, INMT, NOD1 , NR_002186, ZNRF2 chr12: 55685282-56515644 14 0 ARHGAP9, AVIL, B4GALNT1, BC019026, BC033961, BC073932, CDK4, CENTG1, CR625050, CS444342, CTDSP2, CYP1, CYP27B1, DCTN2, DDIT3, DTX3, FAM119B, G43318, GEFT, GLI1, INHBC, INHBE, KIAA0286, KIAA1002, KIF5A, LRP1 , MARCH9, MARS, MBD6, METTL1, MYO1A, NAB2, NDUFA4L2, NXPH4, OS9, PIP5K2C, R3HDM2, RGL1, SHMT2, SLC26A10, STAC3, STAT6 , TAC3, TSFM, TSPAN31, ZBTB39, ZNEUROK1 chr16: 86267434-86992013 14 0 AK126852, AX747795, BANP, CA5A, DKFZp434G0522, FLJ00104, JPH3, KLHDC4, SLC7A5 chr2: 179266611-179839804 13 0 AK123298, CR624402, FLJ39502 , SESTD1, TTN chr3: 95106214-95125111 12 0 PROS1 chr2: 36745429-37640775 11 0 AK001814, BC017652, CCDC75, CEBPZ, EIF2AK2 , HEATR5B, KIAA1414, PRKD3, PRO1853, QPCT, STRN, SULT6B1, VIT chr4: 151504432-151570906 11 0 DKFZp686K03100, LRBA chr11: 127094060-128138864 11 0 AX747861, BC039676, DKFZp686D0662, ETS1 , FLI1 chr1: 31912222-32647867 10 0 AK096192, BAI2, BAI2, BC069257, BC111382, BSDC1, C1orf90, C1orf91, CCDC28B, COL16A1, CR601100, DCDC2B, DKFZp564C2082, DKFZp686B09139, EIF3S2, HDAC1 , IQCC, KHDRBS1 , KPNA6, LCK , LOC339483, MARCKSL1 , PTP4A2, SPOCD1, TMEM39B, TSSK3, TXLNA, UNQ548 chr3: 106447305-107177994 10 0 ALCAM, CBLB, Nbla00127 chr6: 161278798-161959701 10 0 AGPAT4, C6orf59, LPAAT-delta, MAP3K4, MTK1, PARK2 , parkin chr7: 24205709-24955079 10 0 DFNA5, ICERE-1, KIAA0704, MPP6, NPY , OSBPL3 B) Deletions Not Impacting Known Immune Function Genes chr3: 163785970-165636507 94 0 BC073807, LOC647107 chr2: 78159130-78549747 86 0 BC024248, BC030125 chr3: 7053179-7177909 49 0 GRM7 chr11: 109142987-110423998 48 0 AK094117, AK124179, ARHGAP20, DKFZp434I0812, FDX1, KIAA1726, NR_001287, RDX chr12: 27002588-28111983 44 0 AK000807, ARNTL2, BC043511, DKFZp564O1863, FGFR1OP2, KIAA0965, KIAA1230, KLHDC5, LOC728858, MDS023, MRPS35, PPFIBP1, PTHLH, REP15, STK38L, SURB7, TM7SF3 chr6: 97170276-97625708 39 0 C6orf66, FHL5, GPR63, KIAA1900, KLHL32 chr6: 163188894-163315053 35 33591 PACRG chr16: 9624870-10467886 35 0 ATF7IP2, DKFZp666E123, DQ587956, DQ595173, GRIN2A, U07199 chr7: 64220284-64795508 34 0 AK057766, DQ596928, FLJ25037, LOC441242, ZNF92 chr5: 90035491-90386715 33 0 GPR98, KIAA0686, VLGR1 chr3: 37578530-37705809 32 0 ITGA9 chr6: 38413748-38630608 32 0 BTBD9 chr20: 51844911-51931244 28 0 NR_002189 chr15: 57055702-57917444 26 0 BNIP2, CCNB2, DKFZp761D081, FAM81A, GCNT3, GTF2A2, LDHAL6B, MYO1E, RNF111 chr15: 54225066-54550705 24 0 MNS1, TEX9 chr17: 3128134-3194433 23 0 OR3A1, OR3A2, OR3A4 chr7: 7480856-7819889 22 0 AK027125, COL28, COL28A1, FLJ20323, RPA3 chr10: 94222227-94754640 22 0 EXOC6, HHEX, IDE, KIF11 chr14: 91502061-91569101 21 0 TRIP11, Trip230 chr2: 42398844-43092929 19 0 COX7A2L, EML4, HAAO, KCNG3, MTA3, OXER1 chr14: 26535543-27350537 19 0 BX538073 chr14: 92744837-93367366 19 0 BTBD7, C14orf130, COX8C, KIAA1409, PRIMA1 chr2: 14622841-14924411 18 0 AX747684, FAM84A chr12: 10589522-10635183 17 0 KLRA1 chr3: 99229541-99419735 16 0 CR749263, OR5AC2, OR5H1, OR5H14, OR5H15 chr2: 132326867-132449751 15 0 FLJ41821 chr4: 9136858-9881191 15 0 DKFZp586I2219, DQ581767, DQ583133, DQ584082, DQ585713, DQ589421, DRD5, SLC2A9, WDR1 chr4: 128805691-128920529 15 0 INTU, SLC25A31 chr9: 16987947-17478111 15 0 C9orf39, RP11-340N12.1 chr14: 59952568-60075378 15 0 C14orf39, SIX6 chr18: 5682313-5922670 15 0 AF301223, TTMA chr19: 23304120-23389292 15 0 AK022793, BC038574, BC043213, ZNF91 chr12: 39980210-40809427 14 0 GLT8D3, PDZRN4 chr19: 19759149-20368239 14 0 CR593334, CR614976, DKFZp761G18121, FLJ44894, NR_003128, ZNF253, ZNF486, ZNF506, ZNF682, ZNF90, ZNF93 chr2: 98228529-98252357 13 0 DKFZp434J0326, DKFZp451I0318, MGC26733 chr2: 168309981-169429627 13 0 AL080192, B3GALT1, BC035245, LASS6, NOSTRIN, STK39 chr4: 22663503-22803604 13 0 CR607430 chr5: 107695465-109245567 13 0 AK021888, BC034788, FBXL17, FER, MAN2A1, PJA2 chr4: 20984915-21185852 12 0 KCNIP4 chr5: 178315216-178514465 12 0 ADAMTS2, BX648737, CR598488, GRM6, ZNF354C, ZNF454, mGluR6 chr17: 55759021-56436171 12 0 APPBP2, BCAS3, C17orf64, L32131, PPM1D, USP32 chr18: 60137564-60597211 12 0 BC036306 chr5: 38305919-38317501 11 11209 EGFLAM chr6: 12643097-13219417 11 0 PHACTR1, RPEL chr4: 69405358-69494103 10 0 TMPRSS11E chr9: 40497792-40617437 10 0 AK024257 chr11: 102824056-102856185 10 0 DYNC2H1 chr11: 124360372-124420328 10 0 CCDC15 chr12: 47817725-47931507 10 0 TUBA1A, TUBA1B, TUBA1C chr14: 62671250-62971495 10 0 GPHB5, PPP2R5E, RHOJ chr15: 91046103-91378387 10 0 CHD2, DKFZp781D1727, LOC400451 C) Duplications Impacting Immune Function Genes chr19: 5773490-12007370 827 0 ACP5 , ACSBG2, ADAMTS10, AF019226, AF075036, AF161365, AK056073, AK097685, AK124717, ALKBH7, ANGPTL4, ANGPTL6, ANKRD25, ANKRD47, AP1M2, ARHGEF18, ASAH3, ATG4D, AX747405, AX747599, AX748210, AY203940, BC007593, BC014506, BC029904, BC033124, BC039523, BC042816, BGR , C19orf39, C19orf45, C19orf52, C19orf59 , C3 , CAPS, CARM1, CCL25 , CD209 , CD320, CD70 , CDC37, CDKN2D, CLEC4G , CLEC4M , CLPP, CNN1, COL5A3, CR598956, CRB3, CTXN1, DENND1C, DKFZp547H118, DKFZp564K0223, DKFZp564O1762, DKFZp666A071, DKFZp667O2312, DKFZp761J1410, DKFZp761K0816, DNM2, DNMT1 , DOCK6, ECSIT , EDG5, EDG8, EIF3S4, ELAVL1 , ELAVL3, ELOF1, EMR1, EMR4 , EPOR , EVI5L, FBN3, FBXL12, FCER2 , FLJ00153, FLJ11286, FLJ12949, FLJ20079, FLJ22184, FLJ25758, FUT3 , FUT5, FUT6, GPR108, GTF2F1, HNRPM, HSZFP36, ICAM1 , ICAM3 , ICAM4 , ICAM5 , ILF3 , INSR, KEAP1, KHSRP, KIAA0521, KIAA1395, KIAA1518, KIAA1543, KIAA1588, KIAA1776, KIAA1978, LASS4, LDLR, LOC126075, LOC162993, LOC388503, LOC401898, LOC440508, LOC55908, LPPR2, LRRC8E, MAP2K7 , MARCH2, MBD3L1, MBD3L2, MCOLN1, MGC19604, MGC20983, MGC33407, MKK7 , MLLT1, MRPL4, MUC16 , MYO1F, MYO1F variant protein, NDUFA11, NDUFA7, NRTN, OLFM2, OR1M1, OR2Z1, OR7D2, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, P2RY11, PCP2, PDE4A , PEX11G, PIN1, PNPLA6, PPAN, PPAN- P2RY11, PRAM1, PRKCSH, PSPN, QTRT1, RAB11B, RAB3D, RANBP3, RAVER1, RDH8, RETN , RFX2, RGL3, RPS28, SH2D3A, SLC25A23, SLC25A41, SLC44A2, SMARCA4, SNAPC2, SPC24, STXBP2, TIMM44, TMED1 , TNFSF14 , TNFSF9 , TRAPPC5, TRIP10, TSPAN16, TUBB4 , TYK2 , UBL5, UNQ2443, UNQ501, VAV1 , VMAC, XAB2, YIPF2, ZNF121, ZNF177, ZNF266, ZNF317, ZNF358, ZNF414, ZNF426, ZNF433, ZNF439, ZNF440, ZNF441, ZNF491, ZNF557, ZNF558, ZNF559, ZNF560, ZNF561, ZNF562, ZNF627, ZNF653, ZNF69, ZNF699, ZNF700, ZNF763, pp10122 chr14: 92664804-95787550 448 0 AK125038, ASB2, BC016484, BC037859, BC038791, BDKRB2 , BTBD7, BX247990, C14orf109, C14orf130, C14orf132, C14orf139, C14orf142, C14orf152, C14orf48, C14orf49, CLMN, COX8C, CR611440, DDX24, DICER1 , FAM14A, FAM14B, GIG24, GLRX5, GSC, IFI27, KIAA0928 , KIAA1409, KIAA1622, MOAP1, NM_207443, NR_001459, NR_003002, OTUB2, P27, PRIMA1, SERPINA1, SERPINA10, SERPINA11 , SERPINA12, SERPINA13, SERPINA3, SERPINA4, SERPINA5 , SERPINA6 , SERPINA9 , TCL1A , TCL1B, TCL6, TCL6a1, TCL6d1, TML1 chr11: 7678446-10337351 315 0 ADM , AK000908, AK055772, AK090613, ASCL3, AX747224, BC017787, BC027619, BC068088, BC073899, BCA3, C11orf16, C11orf17, CMT4B2, CR598129, DKFZp451C1317, DKFZp686I18166, EIF3S5, IPO7, KIAA0298, KIAA1766, LMO1, MGC10850, NLRP10 , NRIP3, NR_002580, NR_002962, NR_002977, OR10A3, OR10A6, OR5E1P, OR5P2, OR5P3, OVCH2, RAB6IP1, RIC3 , RPL27A, SBF2, SCUBE2, ST5, STK33, SWAP70, TMEM41B, TMEM9B, TUB, U80769, WEE1, ZNF143 chr13: 100600344-103567030 197 0 AK093430, AK096424, AK125748, AX747578, BIVM , C13orf27, CR616826, DKFZp434L2319, ERCC5, FGF14, ITGBL1, KDELC1, LOC196541, RP11-430M15.2- 003, SLC10A2, TPP2, UNQ1910, VGCNL1 chr14: 65357663-66177823 159 0 CR594591, GPHN , MGC88374 chr4: 119628184-120591177 158 0 AK000709, AK024248, AK097701, AK098126, BC035733, BC070391, CEP170L, DQ574659, DQ575011, DQ575856, DQ576410, DQ582480, DQ599872, FABP2 , KIAA0755, KIAA1627, LOC401152, MYOZ2, Myopodin, SEC24D, SYNPO2, USP53 chr8: 106761674-108458326 156 0 ABRA, ANGPT1 , CR602836, Nbla00307, OXR1, ZFPM2 chr4: 113049611-113759418 129 0 ALPK1, C4orf16, C4orf21, C4orf32, CR590073, DKFZp434C0927, FLJ00302, KIAA1527, LOC91431, NEUROG2, TIFA chr15: 40145534-40608559 90 0 CAPN3 , GANC, KIAA0770, PLA2G4D, PLA2G4F, SNAP23, TMEM87A, VPS39, ZFP106 chr14: 43658461-45064383 87 0 AX748292, BC038722, C14orf106, C14orf155, C14orf28, FANCM, FKBP3 , KIAA0423, KLHL28, PRPF39 chr10: 67399581-69536663 75 0 CTNNA3, DNAJC12, HERC4, LRRTM3, MYPN , SIRT1 chr3: 107041527-108030375 71 0 CBLB chr9: 137454076-139460602 70 0 ABCA2, AF161442, AGPAT2, AK023162, AK054908, AK055547, AK090585, AK096249, AK098241, AK128153, AK128864, AL832276, ANAPC2, AX747706, AY952890, BC015688, BC032375, BC034456, BC042667, BC043225, BC061888, BC064596, BC092490, BC101937, BTBD14A, C8G , C9orf116, C9orf139, C9orf140, C9orf142, C9orf163, C9orf167, C9orf75, C9orf86, CAMSAP1, CARD9 , CLIC3, COBRA1, CR614579, CR619051, DKFZp434B205, DKFZp564M173, DKFZp762I052, DPP7, EDF1, EGFL7, ENTPD2, ENTPD8, FAM69B, FBXW5 FLJ20433, FLJ45224, FUT7, GBDR1, GLT6D1, GPSM1, GRIN1, HBE269, INPP5E, KCNT1, KIAA0310, KIAA0649, KIAA1062, KIAA1422, KIAA1984, LCN1, LCN10, LCN12, LCN6, LCN8, LCN9, LHX3, LOC389813, LOC389816, LOC401565, LOC441476, LOC728489, MAMDC4, MAN1B1, MGC14327, MGC59937, MGC61598, MRP- S2, MRPS2, NDOR1, NOTCH1 , NOXA1, NPDC1, NPTIIc, NR1, NR_002958, NR_002975, OBP2A, PAEP , PHPT1, PMPCA, PTGDS, QSCN6L1, RNF208, RP11-100C15.2, RP11-413M3.10, SDCCAG3, SLC34A3, SNAPC4, SOHLH1, SSNA1, TMEM141, TRAF2, TUBB2C, UAP1L1, UBADC1, UNQ2492, UNQ2541, UNQ747, hNMDAR1-3b, pp8875, ve-statin chr18: 73989470-76116152 60 0 AK056304, ATP9B, AX746671, BC016878, BC017654, BC040056, BX537710, C18orf22, CTDP1, DKFZp434A042, KCNG2, NFATC1 , PARD6G, PQLC1, SALL3, TXNL4A, ZNF508, hdim1+ chr9: 134048500-135886847 54 0 ABO, ADAMTS13 , ADAMTSL2, AJ011378, AK123314, AX748058, BARHL1, C9orf166, C9orf7, C9orf9, C9orf96, C9orf98, CEL, CELL, CR592591, CR593670, DBH , DDX31, EEF1A1, FLJ46082, GBGT1, GFI1B, GTF3C4, GTF3C5, KIAA1308, LOC389827, NR_002783, NTNG2, OBP2B, RALGDS , REXO4, RPL7A, SARDH, SETX, SLC2A6 , SURF1, SURF2, SURF4, SURF5, SURF6, TSC1, TTF1 , UNQ2513, VAV2, XPMC2H, vWF-CP chr1: 2182293-3539057 50 0 ACTRT2 , AK021767, AK124865, ARHGEF16, BC114358, C1orf93, CR749717, DQ601700, FAM79A, FLJ42875, HES5, MEGF6, MMEL1 , MORN1, PANK4, PEX10, PLCH2, PRDM16, RER1, RP3-395M20.1, RP3-395M20.10, RP4-740C4.2, SKI, TNFRSF14 , WDR8 chr1: 245537326-245976637 49 0 AB120962, AK130400, BC034303, C1orf150, CIAS1 , NLRP3 , OR13G1, OR2B11, OR2C3, OR2G2, OR2G3, OR2W5, OR5AY1, OR6F1, ZNF496 chr14: 21582956-21976908 47 0 AK093552, AK125397, AV1S3A1T, AV25S1, AV4S1, TCR-[alpha] V 33.1, TCR- alpha, TCRA, TCRAV14.1a, TCRAVN1, TCRD, TRA@, V alpha immunoglobulin , av27s1, hADV14S1, hADV23S1, hADV29S1, hADV38S2, hDV102S1 chr7: 157944418-158676623 46 0 AX746826, BC041429, BC042556, FAM62B, KIAA1228, NCAPG2, PTPRN2, VIPR2 , WDR60 chr19: 48498399-48562727 46 0 CD177 chr22: 34946081-35041308 41 0 APOL1 , APOL2, AX747758, MYH9 chr1: 113185538-113437683 40 0 AFARP1, AX748125, BC023568, BC037540, BC047723, BX648855, LRIG2 , SLC16A1 chr2: 187993955-189286647 39 0 CALCRL, CED-6, GULP1, TFPI chr11: 382079-1386192 38 0 AK094678, AK126635, AP2A2, AX747537, AX748330, BC031953, BC048998, BC066355, BRSK2, C11orf35, CD151, CEND1, CHID1, DEAF1 , DKFZp434K249, DRD4, EFCAB4A, EPS8L2, HRAS , IRF7 , KIAA0899, KIAA1542, LRDD, LRRC56, MG1, MUC5AC, MUC2 , MUC5AC , MUC5B, MUC6, MUCDHL, NR_002585, PDDC1, PEN11B, PKP3, PNPLA2, POLR2L, PTDSS2, RASSF7, RNH1, RPLP2, SCT , SIGIRR , SLC25A22, TALDO1, TMEM16J, TMEM80, TOLLIP , TSPAN4, tollip chr12: 98815353-99473749 37 0 ACTR6, ANKS1B, AX746635, BC048272, BC062763, DEPDC4, DKFZp434M0331, DQ579381, DQ583972, DQ595598, DQ598729, EB- 1, GOLGA2L1, KIAA0701, NR1H4 , SCYL2 , SLC17A8 , hArpX chr4: 7568079-8105372 36 0 ABLIM2, AFAP1, AJ431609, BC043614, KIAA1808, LOC389199, SORCS2 chr14: 23995185-24062459 30 0 AK056368, CMA1 chr14: 103352857-104756910 30 0 ADSS , ADSSL1 , AK057986, AK094143, AKT1 , AX721091, AX746996, BRF, BRF1 , C14orf151, C14orf173, C14orf2, C14orf78, C14orf79, CDCA4, CR602005, DKFZp434N0820, DKFZp434N178, DKFZp686J02145, GPR132 , JAG2, KIAA0284, KIAA0771, KIF26A, LOC374569, LOC400258, MGC23270, NUDT14, PLD4, PPP1R13B, SIVA1, TDRD9, TMEM179, UGPP chr17: 73662981-73765190 23 0 AFMID, BC036810, BIRC5 , EPR-1 , SYNGR2, TK1, UNQ464/PRO809, survivin-3B chr13: 45066410-45918722 22 0 AK095119, AK124928, C13orf18, CPB2, FLJ32682, KIAA0853, LCP1 , LOC220416, LOC283514, RP11-139H14.4-001, RP11-351K3.2-001, SPERT, ZC3H13 chr19: 46758119-46863746 21 0 CEACAM21 , CEACAM4 , UNQ3098 chr8: 6849317-6889488 20 0 AF355799, DEFA3 chr20: 60320976-62223928 17 0 AK056267, AK128329, AL137301, ARFGAP1, ARFRP1, AX747649, AY940852, BC002534, BC025345, BC069708, BC127852, BHLHB4, BIRC7, C20orf11, C20orf135, C20orf149, C20orf151, C20orf166, C20orf195, C20orf20, C20orf58, C20orf59, CABLES2, CHRNA4 , COL20A1, COL9A3, DIDO1, DNAJC5, EEF1A2, FLJ00084, FLJ00118, FLJ30313, GATA5, GMEB2, HRIHFB2281, KCNQ2, KIAA1088, KIAA1269, KIAA1510, LAMA5 , LIME1 , LOC198437, NPBWR2, NR_003244, NR_003245, NTSR1, OATP-E, OGFR, OK/SW-cl.69, 0PRL1, PRIC285, PRPF6, PRR17, PTK6, RGS19, RP4- 697K14.11, RPS21, RTEL1, SAMD10, SLC2A4RG, SLCO4A1, SOX18, SRMS, STMN3, Si- 1-2- 19, TCEA2, TCFL5, TNFRSF6B , TPD52L2, UCKL1, URKL1, YTHDF1, ZBTB46, ZGPAT, ZNF512B chr2: 86924985-87026840 16 0 CD8B , RGPD1, RMND5A chr6: 128245227-128538250 16 0 C6orf190 , PTPRK chr1: 111628132-111661435 15 0 CHIA , RP11-165H20.1 chr7: 80041654-80110504 13 0 CD36 chr12: 6085895-6134080 11 0 VWF chr12: 17854127-19338878 11 0 CAPZA3 , FLJ22655, PIK3C2G, PLCZ1, PLEKHA5 D) Duplications Not Impacting Known Immune Function Genes chr8: 430424-977192 189 0 AK128400, BC022082, BC038783, C8orf42, DQ584928, ERICH1, LOC389607 chr10: 2682656-3123648 157 0 PFKP chr19: 21575835-22802826 152 0 BC030765, CR936832, FKSG70, ZNF100, ZNF208, ZNF257, ZNF43, ZNF492, ZNF676, ZNF99 chr6: 63118894-64091007 116 0 GLULD1, LGS chr2: 126935385-127317174 98 0 GYPC chr5: 169763949-170518986 96 0 DKFZp666P032, GABRP, KCNIP1, RANBP17 chr6: 1977258-2424512 92 0 AJ420566, AK023629, AK091028, CR598484, GMDS chr1: 145196592-145539979 65 0 BC036212, BCL9, CHD1L chr3: 62813828-63789968 63 0 BC043407, C3orf49, CADPS, FLJ44379, SYNPR chr5: 172824934-173106866 62 0 BC033564, FAM44B chr3: 83912925-84849961 54 0 BC068246 chr11: 106523381-106702850 54 0 CWF19L2 chr7: 50911090-51127086 50 0 COBL chr9: 6557841-7007391 41 0 AK098534, BC042976, GLDC, JMJD2C, KIAA0780 chr17: 793231-917163 40 0 ABR, NXN, TIMM22 chr3: 148425802-149211699 39 0 AK098763, ZIC1, ZIC4 chr13: 62536330-64097876 36 0 AK057471, AK097490, AK098560, BC128161, NR_002171 chr4: 92669000-94351161 34 0 GRID2, KIAA1680 chr3: 11041670-11154453 33 0 SLC6A1 chr4: 141709869-141939520 33 0 TBC1D9 chr13: 113032851-113140550 30 0 ADPRHL1, GRTP1 chr2: 148947731-149053581 29 0 KIAA1461, MBD5 chr3: 141649121-141911988 28 0 CLSTN2, TRIM42 chr12: 20901315-21459060 27 0 IAPP, LST-3TM12, LST3, SLCO1A2, SLCO1B1, SLCO1B3 chr12: 130676799-131128244 27 0 EP400, KIAA1498, KIAA1818, MMP17, NR_002979, PUS1, SFRS8, ULK1 chr7: 34919485-35862821 26 0 AJ011981, BC049371, BC084560, CR593784, CR595224, DPY19L1, DPY19L2P1, HERPUD2, KIAA0877, SEPT7, T8X20 chr1: 30940148-31013899 25 0 BC044253, LAPTM5, MATN1 chr4: 63433227-65099386 25 0 SRD5A2L2 chr5: 12838251-13150705 22 0 AY328033 chr6: 138653005-138759016 22 0 KIAA1244 chr1: 103111860-103157198 21 0 COL11A1 chr6: 34836231-34976532 21 0 ANKS1A, C6orf107, SNRPC, TAF11 chr15: 27167056-28188067 21 0 APBA2, BC043570, BC070492, BC071630, BC071855, DQ572986, DQ573498, DQ575284, DQ575742, DQ577333, DQ578370, DQ578838, DQ582641, DQ582940, DQ590322, DQ592322, DQ595055, DQ596303, DQ596319, DQ597873, KIAA0574, NDNL2, TJP1, hXIIL chr2: 3287465-3327227 20 0 TSSC1 chr10: 98101269-98152781 19 0 TLL2, TMEM10 chr1: 79037280-79973037 18 0 ELTD1 chr4: 68749536-70089270 18 0 AK123556, TMPRSS11B, TMPRSS11E, UGT2A3, UGT2B10, UGT2B15, UGT2B17, UGT2B7, YTHDC1 chr2: 27584444-28424525 17 0 AK124439, BC041993, BC048132, BRE, C2orf16, CCDC121, GCKR, MRPL33, RBKS, SLC4A1AP, SUPT7L, XAB1, ZNF512 chr2: 172356838-172528561 17 0 HAT1, SLC25A12 chr2: 180123158-180216026 16 0 ZNF533 chr4: 15529521-15552238 15 0 FGFBP1 chr18: 14805527-14905835 15 0 ANKRD30B chr5: 1601764-1621442 14 0 CR749689 chr10: 88722456-89266679 14 0 AK091716, BC036645, BC047063, BC065757, BC082979, BC092519, CR609725, CR614919, FAM35A, GLUD1, KIAA1975, KIAA2020, MINPP1 chr1: 225308870-225407413 13 0 CDC42BPA chr7: 74674968-74738702 13 0 LOC441257, PMS2L14 chr1: 193808000-194531039 12 0 KCNT2, SLICK chr2: 165344115-165435694 12 0 COBLL1, KIAA0977 chr6: 123847128-124726881 12 0 TCBA1, TRDN chr16: 26961662-26994169 12 0 TNT chr6: 102029392-102048325 11 11862 GRIK2 chr8: 11397047-11414199 11 0 BLK chr8: 27689983-27745887 11 0 ESCO2, PBK chr2: 77709215-78761206 10 0 BC024248, BC030125 chr11: 5444172-5454375 10 11939 HBE1, HBG2, OR51B5 chr11: 19571323-19595111 10 96377 NAV2 chr18: 68553733-69070964 10 0 BC013370, BC034583, NETO1 chr19: 34552727-34574348 10 0 AK094793 chr4: 2059850-2960297 10 0 AB000464, AB000465, AB000466, ADD1, AK054619, BC010180, BC032331, C4orf10, C4orf15, C4orf8, CR622423, GRK4, KIAA1643, MXD4, Mad4, NOL14, POLN, RNF4, SH3BP2, TETRAN, TNIP2, ZFYVE28

TABLE 5 Homozygous Deletions Observed Exclusive to Cases Cases Distance From CNVR Loss Gene Distance Exon chr3: 100430538-100430538 2 CLCP1, DCBLD2 327315 327315 chr2: 118777060-118778863 1 INSIG2 192993 192993 chr3: 146130742-146141275 1 DQ595575 883692 883692 chr5: 29455914-29488308 1 AK098570 247692 247692 chr5: 141998044-142000692 1 FGF1 0 16953 chr6: 32525108-32530999 1 HLA-DRA 4306 4306 chr6: 62122152-62144592 1 G43499 251536 251536 chr6: 77159914-77159914 1 IMPG1 320859 320859 chr6: 128018101-128024702 1 C6orf190 46336 46336 chr12: 17277311-17282429 1 LMO3, Nbla03267 625020 625020

TABLE 6 Genome Global CNV Burden Significant Including Common CNV Exonic Locus Locus Exonic Locus Non- Exonic Locus Non- Significant Exonic Significant Exonic Case Case Control Control Enriched Enriched Enriched Enriched P DifFreq Deletion 21 61 8 67 0.022628 0.149431 Duplication 43 173 12 12 0.003336 −0.30093 Case CNV Control Case CNV Calls Non- Control CNV CNV Calls Calls Exonic Exonic Calls Exonic Non-Exonic P DifFreq Rare CNV Exonic Deletion 527 1149 3970 10290 0.002138 0.036038 Duplication 813 1785 5539 5997 2.45E−55 −0.16722 Rare CNV >100 KB Exonic Deletion 136 83 1012 653 0.768312 0.013197 Duplication 278 146 2263 546 2.46E−11 −0.14996 Samples w Rare CNV >100 KB (Impacting gene or not) Case Control Case with without Control with without large rare large rare large rare large rare CNV CNV CNV CNV P DifFreq Deletion 131 180 1156 1610 0.951669 0.00329 Duplication 186 125 1391 1375 0.001504 0.095178

Finally, to address the heterogeneity of the CVID phenotype and comorbid clinical manifestations that affect subsets of patients, we compared CVID patients with common sub-phenotypes to patients without any additional comorbidities. Based on recent phenotypic characterization²², we established 16 distinct clinical subtracks within CVID to study: cancer, lymphoma, lymphadenopathy, nodular regenerative hyperplasia of the liver (NRH), lymphoid interstitial pneumonitis (LIP), bronchiectasis, biopsy proven granuloma, GI enteropathy, malabsorption, splenectomy, cytopenias, organ specific autoimmunity (OSAI), low IgM (<50 mg/dL), low IgA (<10 mg/dL), low B cells (CD19+ cells <1%), and young age of symptom onset (<10 yrs) (FIG. 5 and Table 7). The case and control labels within the CVID cohort were based on previously defined criteria. Differences in genotype frequencies were assigned significance and regions with multiple significant SNPs were then scored. Significant SNP associations were made for all subsets (Table 8 and FIG. 1C). The most significant association was observed with NRH on AK096081-AK124028 (P=2.29×10⁻¹⁰). Lymphoma was associated with KIAA0834, PFTK1 and HAVCR1, with corresponding P values ranging from P=1.69×10⁻⁸-3.62×10⁻⁸. An association was also observed between LIP and FGF14 and ZNF81, with corresponding P values of P=5.76×10⁻⁸ and P=7.70×10⁻⁸, respectively. OSAI also associated with SNX31 (P=6.89×10⁻⁸) and low IgM levels (<50 mg/dL) in the case subjects were associated with LDLRAP1 (P=6.02×10⁻⁸). Patients with CVID manifesting enteropathy and their resulting subphenotype significance were additionally queried against the IBD significance observations to assess if specific loci contributed to a common etiology. The SNP rs12889533 was significant in both CVID+enteropathy and IBD with allele frequency difference in the same direction (Table 9).

TABLE 7 CVID Subphenotypes and Sample Sizes CVID Subphenotype Cases Controls Cancer 13 249 Lymphoma 9 253 Lymphadenopathy 30 232 NRH 13 249 LIP 10 252 Bronchiectasis 78 184 Granuloma 34 228 GIEnteropathy 19 243 Malabsorption 13 249 Splenectomy 40 222 Cytopenias 27 235 OSAI 75 187 Low IgM (<50 mg/dL) 206 56 Low IgA (<10 mg/dL) 160 102 CD19 (<1%) 11 251 AgeOnsetSx (<10 yrs) 42 220

TABLE 8 Most Significant Associated Regions Based on Subphenotype Genotype Association Significant Count Distance Region P-value SNP A1 F_A F_U OR SNPs Gene Distance From Exon Subphenotype chr1: 68153970- 2.29E−10 rs1926283 C 0.2308 0.01807 16.3 3 AK096081, 0 33408 NRH 68161019 AK124028 chrX: 132092989- 5.50E−10 rs5977837 C 0.2778 0.02174 17.31 2 TFDP3 82912 82912 Lymphoma 132095451 chr10: 58755852- 1.36E−09 rs16910534 T 0.2222 0.01383 20.37 3 IPMK 858286 858286 Lymphoma 58767334 chr20: 55901032- 3.21E−09 rs8124301 T 0.4231 0.07631 8.877 5 C20orf85 174030 174030 Malabsorption 55985359 chr7: 90245135- 1.69E−08 rs975004 G 0.3333 0.03953 12.15 2 KIAA0834, 0 12544 Lymphoma 90245280 PFTK1 chr5: 156402979- 3.62E−08 rs10038271 T 0.7222 0.1877 11.25 2 HAVCR1 0 651 Lymphoma 156407976 chrX: 22422160- 5.71E−08 rs5925651 A 0.3462 0.1386 3.291 3 ZNF645 219665 219665 Bronchiectasis 22428888 chr13: 101641990- 5.76E−08 rs1336698 G 0.65 0.168 9.197 6 FGF14 0 69139 LIP 101673539 chr1: 25735669- 6.02E−08 rs2065970 G 0.06553 0.2411 4.529 5 LDLRAP1 0 0 Low IgM 25753653 chrX: 143116309- 6.77E−08 rs6649722 A 0.3 0.0377 10.94 3 UBE2NL 320286 320286 LIP 143119321 chr8: 101728102- 6.89E−08 rs7815950 G 0.2133 0.05615 4.559 2 SNX31 0 2334 OSAI 101728344 chr8: 13834782- 7.62E−08 rs2682665 C 0.1818 0.01394 15.71 3 SGCZ 149368 149368 Low B cells 13842376 chrX: 47642545- 7.70E−08 rs12387999 A 0.2 0.016 15.38 2 ZNF81 0 2262 LIP 47651320 chrX: 152295068- 9.84E−08 rs5987017 A 0.2143 0.04773 5.442 4 ZNF275 24829 24829 Young age 152303019 chr12: 2531014- 1.21E−07 rs4765961 C 0.4737 0.142 5.439 3 CACNA1C 0 0 GI Enteropathy 2538733 chrX: 30733771- 1.44E−07 rs11095197 T 0.1469 0.3431 3.034 2 MAP3K7IP3 18876 18876 Low IgA 30736604 chrX: 37803525- 1.54E−07 rs5918500 C 0.4878 0.2123 3.533 3 SYTL5 0 0 Young age 37832251 chrX: 53035117- 1.90E−07 rs10127016 G 0.3833 0.125 4.351 2 TMEM29 0 1409 Lymphade- 53039801 nopathy chr7: 107306707- 2.16E−07 rs9690688 A 0.3636 0.06375 8.393 2 DLD 4709 4709 Low B cells 107314113 chr21: 18188480- 2.84E−07 rs7280675 G 0.1923 0.02008 11.62 2 CHODL 2636 2636 NRH 18192815 chr1: 55704352- 2.85E−07 rs356086 G 0.1111 0.00395 31.5 2 FLJ45337 112975 112975 Lymphoma 55706323 chr21: 45476504- 3.23E−07 rs4592938 A 0.1154 0.00602 21.52 2 C21orf89 1777 1777 NRH 45476918 chr14: 23168471- 4.57E−07 rs222723 C 0.6154 0.1968 6.531 2 DHRS2 0 0 Cancer 23169215 chr2: 109118795- 4.62E−07 rs375099 C 0.25 0.02976 10.87 2 POSH2 0 5794 LIP 109120561 chr12: 30583074- 4.82E−07 rs1905675 C 0.2 0.5453 0.209 2 IPO8 74732 74732 Lymphade- 30598457 nopathy chrX: 39933689- 5.18E−07 rs2948491 A 0.18 0.04545 4.61 4 BCOR 12163 12163 OSAI 39956544 chr14: 64723761- 8.09E−07 rs4299072 A 0.1667 0.02802 6.938 4 BX161428 0 0 Lymphade- 64783067 nopathy chr7: 117377201- 9.94E−07 rs17140937 C 0.2692 0.04418 7.971 2 CTTNBP2 76404 76404 Malabsorption 117382435 OR: Odds Ratio Additional detail in Table 13.

TABLE 9 CVID Patients with GI Enteropathy vs CVID Patients without Genotype Association Replication in IBD Based on Single SNP Sigificance P_CVID + SNP Gene Distance GIEnteropathy(IBD) Allele1 F_A F_U OR P_IBD Allele1 F_A F_U OR rs6532122 GPRIN3 3497 0.001204 A 0.2632 0.0947 3.42 9.90E−8 A 0.0786 0.1072 0.7109 rs17041264 COLQ 196 0.000368 T 0.1579 0.0350 5.17 3.15E−6 T 0.0049 0.0135 0.3608 rs12889533 C14orf143 19903 0.000561 T 0.1316 0.4156 0.21 1.28E−4 T 0.3563 0.3878 0.8736 rs12889533 replicates with allele frequency in the same direction while rs6532122 and rs17041264 are significant in the opposite direction of allele frequency.

Finally, to identify potential functional biases specific to CVID, we evaluated clustering into specific INTERPRO categories using DAVID²³: Database for Annotation, Visualization, and Integrated Discovery. We found two significant INTERPRO categories with genes contributing from several of our different analysis methods. First, “Interleukin 1/heparin-binding growth factor” P=9.60×10⁻³ including FGF1, FGF23, FGF6, and FGF14 from subphenotype analysis of lymphadenopathy, cytopenias, LIP, and low IgA. Secondly, “Immunoglobulin I-set” P=5.40×10⁻² including CHL1, CNTN4, LINGO2, SDK1 from overall GWAS analysis, cytopenias subphenotype, and duplication CNV.

Discussion

CVID as a clinical phenotype was described more than 50 years ago, but aside from a small number of recessively inherited genes in a few families, and the more prevalent but poorly understood contribution of mutations in TNFRSF13B^(5,6,7,24), other causes have remained obscure. CVID has thus been hypothesized to represent a diverse collection of genetic lesions resulting in a similar immunologic phehotype. The MHC region has been associated with a myriad of complex diseases²⁵ including immune-related conditions²⁶ and CVID^(8,9). MHC was robustly associated with this CVID in our study, confirming the prior work that was not performed at the genome-wide level. However, the SNP genotype association analysis presented here also revealed novel genes, with the most significant CVID associations outside of the MHC region with a locus encompassing ADAM28, ADAM7, ADAMDEC1 and STC1 (P=6.24×10⁻⁶). The ADAM family proteins are zinc metalloproteases involved in diverse biologic processes, including immune responses. Interestingly, the metallopeptidase MMP27 was also associated with CVID (P=1.69×10⁻⁴). Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix to promote routine physiological processes, but may also facilitate disease pathogenesis. Related genes have demonstrated immunological function involved in the regulation of cytokine release, Th2 immune responses and specific inflammatory processes. ADAM28 is also known as the lymphocyte metalloprotease MDC-L, expressed on the lymphocyte cell surface²⁷. As such, it has been defined as a ligand for α4β1 integrin which enables the adhesion of other leukocytes expressing this integrin. STC1 in the same region, is a gene that encodes stanniocalcin 1 protein, involved in regulation of calcium including in antioxidant pathways of marcophages²⁸. In this light, calcium regulation within immune cells has been previously identified as aberrant in certain CVID patient samples²⁹. UBX10 may be a compelling candidate potentially involved in immunopathogenesis of primary antibody failure in that it encodes an Ubiquitin-like protein, which in addition to phosphorylation, has been shown to regulate NF-κB activity³⁰. SDK1 (Sphingosine-dependent protein kinase-1) is important in the survival of alveolar macrophages³¹. DEPDC6 is a negative regulator of mTORC signaling pathways and RNA expression levels were found to be significantly different between those mice resistant to H5N1 influenza virus when compared to those that were susceptible³². No significant associations in the region of TACI were observed, and subjects with TACI mutations were not separately identified. We reviewed the reported amino acid changes and looked up their corresponding non-synonymous SNP IDs (Table 10). No imputation reference files including these SNPs were available to infer genotypes.

TABLE 10 TACI Protein TNFRSF13B Gene Amino Acid Variant and Corresponding nsSNP rs ID Clinical Report nsSNP DNA position Alleles AA Change (bold-in reports) rs34562254 16783716 C/T P (CCC) --> L (CTC) P251L rs72553886 16783732 G/T V (GTC) --> F (TTC) V246F rs56063729 16783809 A/G V (GTG) --> A (GCG) V220A rs56248318 16784408 A/C Q (CAG) --> H (CAT) Q196H rs72553885 16784417 A/C C (TGC) --> * (TGA) C193X rs72553883 16784454 A/C A (GCG) --> E (GAG) A181E rs72553882 16784504 A/C Y (TAC) --> * (TAA) Y164X rs72553881 16784541 A/G G (GGG) --> E (GAG) G152E rs72553880 16792777 A/G A (GCT) --> T (ACT) A149T rs72553879 16792911 A/G C (TGT) --> Y (TAT) C104Y rs34557412 16792912 C/T C (TGT) --> R (CGT) C104R rs72553877 16792962 A/T I (ATC) --> N (AAC) I87N rs72553876 16792986 A/G Y (TAT) --> C (TGT) Y79C rs55916807 16793007 C/T R (CGC) --> H (CAC) R72H rs67951770 16796563 C/G D (GAT) --> H (CAT) D41H rs67951769 16796563 —/G frameshift D41fx rs72553874 16796566 C/T W (TGG) --> R (CGG) W40R rs72553884 16784425-16784424 —/G Frameshift D191G rs72553878 16792925-16792924 —/T Frameshift Q99H rs34182967 16793004-16793003 —/C Frameshift K73R rs72553875 16793018-16793017 —/A Frameshift S68S

TABLE 11 Most Significant Associated Regions Based on Genotype Association Count SNPs Distance Region Top From Genomic Span Gene 1000 Exon BestP BestSNP Allele1 F_A F_U chr6: 27236785- MHC 110 0  8.62E−10 rs3117426 T 0.3268 0.1907 32521295 chr8: 23746576- ADAM28, 4 0 6.24E−6 rs4872262 A 0.0391 0.0107 24681608 ADAM7 , ADAMDEC1, STC1 chr4: 189671181- AK095968 2 18764 1.78E−5 rs1606234 T 0.2961 0.4124 189676119 chr10: 73083830- CDH23 2 7287 2.45E−5 rs7087554 A 0.5894 0.4729 73084583 chr7: 4270929- SDK1 5 0 4.70E−5 rs895710 T 0.2905 0.4003 4287047 chr1: 20369369- FLJ32784, 7 0 9.09E−5 rs7514144 C 0.2654 0.1808 20490791 UBXN10 chr10: 27709830- PTCHD3 7 0 9.77E−5 rs506659 T 0.4022 0.3026 27728115 chr1: 59987116- FGGY 2 2458 1.52E−4 rs11207520 G 0.3994 0.3026 59993733 chr12: 80435676- PPFIA2 2 59900 1.63E−4 rs2400955 T 0.4134 0.3159 80444630 chr11: 102069786- MMP27 2 0 1.69E−4 rs17099394 T 0.1229 0.0687 102079454 chr16: 71592439- ZFHX3 2 40845 1.80E−4 rs8056528 C 0.3687 0.2754 71594134 chr8: 124729628- KLHL38 4 0 2.05E−4 rs4871402 A 0.0643 0.1325 124734756 chr3: 7364204- GRM7 4 40865 2.96E−4 rs12491592 A 0.1536 0.0939 7369287 chr3: 8378739- BC020876 2 129448 3.51E−4 rs359030 C 0.1648 0.1033 8381919 chr6: 25570803- LRRC16A 2 0 3.61E−4 rs4320355 T 0.3799 0.2898 25574868 chr6: 153354039- MTRF1L 2 0 3.81E−4 rs9322400 T 0.4469 0.3526 153355207 chr3: 284363- CHL1 2 19083 4.31E−4 rs17273893 T 0.148 0.0908 285747 chr8: 121001793- DEPDC6 2 0 4.41E−4 rs869340 C 0.324 0.2402 121010709 chr2: 65518304- FLJ16124 2 784 5.20E−4 rs1194849 G 0.3659 0.4614 65520560 chr21: 27713547- BC043580 2 19690 5.51E−4 rs469709 A 0.1704 0.1095 27722878 P F_A F_U Replication Replication Replication BestP BestSNP F_A F_U Region (Discovery (Discovery (Discovery Repli- Repli- Repli- Repli- Genomic Span SNP) SNP) SNP) cation cation cation cation chr6: 27236785- 0.3634 0.1697 0.1468 0.0004 rs2156875 0.5734 0.4475 32521295 chr8: 23746576- 0.03135 0.0321 0.01346 0.0314 rs4872262 0.0321 0.0135 24681608 chr4: 189671181- 0.4646 0.4266 0.4524 0.4646 rs1606234 0.4266 0.4524 189676119 chr10: 73083830- 0.4591 0.5092 0.4829 0.4591 rs7087554 0.5092 0.4829 73084583 chr7: 4270929- 0.4551 0.3578 0.3835 0.0235 rs4720301 0.3991 0.4793 4287047 chr1: 20369369- 0.3018 0.1560 0.1311 2.25E−8 rs6426636 0.4679 0.2857 20490791 chr10: 27709830- 0.9879 0.2798 0.2793 0.6291 rs493965 0.2844 0.3001 27728115 chr1: 59987116- 0.00148 0.2569 0.1706 0.0015 rs11207520 0.2569 0.1706 59993733 chr12: 80435676- 0.8687 0.3899 0.3842 0.5347 rs10746192 0.4306 0.4526 80444630 chr11: 102069786- 0.6947 0.0734 0.06643 0.6947 rs17099394 0.0734 0.0664 102079454 chr16: 71592439- 0.3698 0.3670 0.3368 0.3698 rs8056528 0.367 0.3368 71594134 chr8: 124729628- 0.7201 0.1009 0.1088 0.3161 rs7463896 0.1697 0.1979 124734756 chr3: 7364204- 0.5051 0.0963 0.08318 0.1237 rs7617297 0.3073 0.2592 7369287 chr3: 8378739- 0.1446 0.1147 0.08535 0.1139 rs358994 0.1055 0.0754 8381919 chr6: 25570803- 0.2219 0.2844 0.2469 0.1896 rs301396 0.445 0.3993 25574868 chr6: 153354039- 0.454 0.3945 0.3688 0.454 rs9322400 0.3945 0.3688 153355207 chr3: 284363- 0.6342 0.0963 0.08678 0.6342 rs17273893 0.0963 0.0868 285747 chr8: 121001793- 0.7402 0.3073 0.2966 0.682 rs4871793 0.3113 0.2978 121010709 chr2: 65518304- 0.01381 0.3899 0.4771 0.0119 rs1876518 0.3853 0.4744 65520560 chr21: 27713547- 0.9615 0.0963 0.09532 0.6677 rs17631106 0.0688 0.0769 27722878 Bold underline: Known immunological function Gene

TABLE 12 Most Significant Associated Regions Based on CNV Association A) Deletions Distance CNVR Count From P Cases Controls Com- Deletion SNPs Exon Deletion Del Del Gene MSSM Oxford USF bined chr11: 85365857- 5 0 0.01 2 0 PICALM , PICALM variant protein 0 2 0 2 85381622 chr20: 57735790- 6 5902 0.026 3 4 PHACTR3 2 1 0 3 57741780 chr22: 17396663- 270 0 0.029 2 1 ARVCF, C22orf25, C22orf29, 2 0 0 2 18417315 CDC45L, CLDN5, CLTCL1, COMT , CR618542, CR625276, DGCR14, DGCR2, DKFZp761P1121, DKFZp781E0833, GNB1L, GP1BB, GSCL, HIRA, KIAA1647, L77561, LOC128977, MRPL40, SEPT5, SLC25A1, TBX1 , TRXR2A, TSSK2, TXNRD2, U84523, UFD1, UFD1L chr4: 10256682- 4 2915 0.029 2 1 CLNK 0 1 1 2 10264316 chr10: 46003146- 8 0 0.029 2 1 DKFZp566K0524, PTPN20A, 2 0 0 2 46042543 PTPN20B B) Duplications Distance CNVR Count From Cases Controls Com- Duplication SNPs Exon P Dup Dup Dup Gene MSSM Oxford USF bined chr2: 148396730- 6 0 8.66E−16 15 0 ACVR2A , ORC4L 10 5 0 15 148433180 chr15: 35053039- 6 23146 5.72E−13 19 10 MEIS2 17 2 0 19 35063531 chr10: 53044735- 4 79826 9.63E−13 12 0 PRKG1 12 0 0 12 53045426 chr8: 77719805- 3 27377 1.05E−09 10 1 BC037827 10 0 0 10 77720240 chr7: 91789778- 4 0 1.20E−09 12 4 ANKIB1 9 3 0 3 91801963 chr2: 163316298- 3 17100 1.00E−07 7 0 KCNH7 6 1 0 7 163316595 chr7: 87180702- 4 202 7.41E−07 7 1 RPIB9 5 2 0 7 87191931 chr12: 72825720- 3 0 3.04E−06 7 2 BC061638 7 0 0 7 72832667 chr2: 203004035- 5 20466 6.58E−05 6 3 BMPR2 6 0 0 6 203017311 chr19: 9256584- 4 0 0.001 3 0 ZNF699 2 0 1 0 9277749 chr4: 39190766- 6 0 0.004 3 1 UGDH 1 2 0 3 39201960 chr9: 112207473- 3 553 0.004 3 1 SVEP1 3 0 0 3 112207708 chr7: 18903915- 4 23171 0.009 3 2 HDAC9 1 2 0 3 18905725 chr13: 64053258- 15 505114 0.01 2 0 AK057471, AK098560, BC128161 2 0 0 2 64056530 chr14: 35408995- 5 0 0.01 2 0 BRMS1L 0 2 0 2 35422074 chr5: 170531269- 4 374 0.01 2 0 DKFZp666P032, RANBP17 1 1 0 2 170536993 chr4: 91413829- 3 33381 0.01 2 0 KIAA1680, MGC48628 2 0 0 2 91415037 chr7: 34685030- 4 4517 0.01 2 0 AAA1, NPSR1 1 1 0 2 34686172 chr3: 176427638- 7 5118 0.01 2 0 NAALADL2 2 0 0 2 176429297 chr1: 170400944- 4 26274 0.01 2 0 DNM3 0 1 1 2 170403742 chr2: 167031393- 4 0 0.01 2 0 Na+ channel, SCN7A 2 0 0 2 167045981 chr9: 28683005- 3 21420 0.01 2 0 LINGO2 2 0 0 2 28687679 chr9: 105860023- 9 27434 0.01 2 0 OR13C3, OR13C4, OR13C5, OR13C8, 2 0 0 2 105868928 OR13F1, SMC2, hCAP-E

TABLE 13 Most Significant Associated Regions Based on Subphenotype Genotype Association Distance Count From Significant Region P-value SNP A1 F_A F_U OR SNPs Gene Distance Exon A) Cancer chr14: 23168471- 4.57E−7 rs222723 C 0.6154 0.1968 6.531 2 DHRS2 0 0 23169215 chr6: 140225308- 1.53E−6 rs12111348 T 0.4231 0.1064 6.157 2 BC039503 2009 2009 140225386 B) Lymphoma chrX: 132092989-  5.50E−10 rs5977837 C 0.2778 0.02174 17.31 2 TFDP3 82912 82912 132095451 chr10: 58755852- 1.36E−9 rs16910534 T 0.2222 0.01383 20.37 3 IPMK 858286 858286 58767334 chr7: 90245135- 1.69E−8 rs975004 G 0.3333 0.03953 12.15 2 KIAA0834, PFTK1 0 12544 90245280 chr5: 156402979- 3.62E−8 rs10038271 T 0.7222 0.1877 11.25 2 HAVCR1 0 651 156407976 chr1: 55704352- 2.85E−7 rs356086 G 0.1111 0.003953 31.5 2 FLJ45337 112975 112975 55706323 chr1: 106430611- 1.55E−6 rs11184786 T 0.3333 0.05336 8.87 2 BC043293 467531 467531 106438079 C) Lymphadenopathy chrX: 53035117- 1.90E−7 rs10127016 G 0.3833 0.125 4.351 2 TMEM29 0 1409 53039801 chr12: 30583074- 4.82E−7 rs1905675 C 0.2 0.5453 0.2085 2 IPO8 74732 74732 30598457 chr14: 64723761- 8.09E−7 rs4299072 A 0.1667 0.02802 6.938 4 BX161428 0 0 64783067 chr5: 141916656- 1.13E−6 rs17706715 A 0.2333 0.05603 5.127 2 FGF1 27191 27191 141926115 chr11: 101265604- 1.55E−6 rs17097290 T 0.15 0.02371 7.267 2 ANGPTL5 513 513 101266102 D) Nodular regenerative hyperplasia of the liver (NRH) chr1: 68153970-  2.29E−10 rs1926283 C 0.2308 0.01807 16.3 3 AK096081, AK124028 0 33408 68161019 chr21: 18188480- 2.84E−7 rs7280675 G 0.1923 0.02008 11.62 2 CHODL 2636 2636 18192815 chr21: 45476504- 3.23E−7 rs4592938 A 0.1154 0.006024 21.52 2 C21orf89 1777 1777 45476918 chr8: 143046590- 1.18E−6 rs12676273 G 0.3462 0.07229 6.794 3 TSNARE1 235455 235455 143055893 chrX: 27453340- 1.29E−6 rs5971431 C 0.3077 0.05823 7.188 3 AK057304 55692 55692 27462759 E) LIP chr13: 101641990- 5.76E−8 rs1336698 G 0.65 0.168 9.197 6 FGF14 0 69139 101673539 chrX: 143116309- 6.77E−8 rs6649722 A 0.3 0.0377 10.94 3 UBE2NL 320286 320286 143119321 chrX: 47642545- 7.70E−8 rs12387999 A 0.2 0.016 15.38 2 ZNF81 0 2262 47651320 chr2: 109118795- 4.62E−7 rs375099 C 0.25 0.02976 10.87 2 POSH2 0 5794 109120561 chrX: 30122360- 2.52E−6 rs5927496 C 0.7 0.2341 7.633 2 MAGEB2 17927 17927 30125674 F) Bronchiectasis chrX: 22422160- 5.71E−8 rs5925651 A 0.3462 0.1386 3.291 3 ZNF645 219665 219665 22428888 chr2: 200959722- 2.39E−6 rs13019534 G 0.5064 0.2908 2.503 2 AOX1, DNAPTP6, 0 0 201175137 KCTD18, SGOL2 chr8: 15782386- 2.92E−6 rs1563297 A 0.5321 0.3152 2.47 2 TUSC3 116020 116020 15788801 chr19: 51765478- 6.30E−6 rs6509286 C 0.4167 0.2228 2.491 2 AK094504 0 27862 51768118 chrX: 68569536- 1.04E−5 rs7056340 A 0.3141 0.1467 2.663 2 TMEM28 68076 68076 68573727 chr6: 32508322- 1.12E−5 rs2027856 T 0.02564 0.163 0.1351 2 HLA-DRA 4942 4942 32510683 G) Granuloma chr11: 12182328- 4.83E−6 rs16910765 T 0.2206 0.05921 4.497 3 KIAA0750, MICAL2, 0 0 12186013 MICAL2PV1, MICAL2PV2 chr1: 33674005- 1.26E−5 rs12751162 A 0.3824 0.1601 3.248 2 PHC2 4803 4803 33683227 chr6: 88453628- 2.04E−5 rs2250276 G 0.1765 0.04386 4.671 2 AKIRIN2 0 5428 88454474 chrX: 27453340- 3.18E−5 rs5971431 C 0.1912 0.05263 4.255 2 AK057304 55692 55692 27462759 H) GI Enteropathy chr12: 2531014- 1.21E−7 rs4765961 C 0.4737 0.142 5.439 3 CACNA1C 0 0 2538733 chr10: 26643326- 2.90E−6 rs7903552 G 0.3684 0.107 4.869 2 GAD2 9833 9833 26646318 chr5: 11592622- 6.26E−6 rs2727602 T 0.6053 0.2613 4.334 2 CTNND2 0 21030 11597026 chr21: 42551877- 9.95E−6 rs3787986 T 0.3684 0.1152 4.479 4 ABCG1 0 0 42552623 chr9: 4033590- 1.13E−5 rs676472 C 0.2895 0.07613 4.944 3 GLIS3 0 68391 4039110 chr4: 33014145- 1.43E−5 rs6846113 A 0.1053 0.01029 11.32 2 AK093205 552926 552926 33017023 I) Malabsorption chr20: 55901032- 3.21E−9 rs8124301 T 0.4231 0.07631 8.877 5 C20orf85 174030 174030 55985359 chr7: 117377201- 9.94E−7 rs17140937 C 0.2692 0.04418 7.971 2 CTTNBP2 76404 76404 117382435 chr11: 124071934- 1.25E−6 rs1784539 G 0.6154 0.2068 6.136 2 SPA17 2037 2037 124079438 chr10: 30255454- 5.18E−6 rs11007812 T 0.3077 0.06426 6.472 2 CR626438 85918 85918 30255817 chr12: 31276546- 6.76E−6 rs12819069 C 0.3846 0.09839 5.727 2 OVOS2 26191 26191 31281817 chr8: 6374232- 8.56E−6 rs2515477 T 0.4231 0.1185 5.456 2 ANGPT2 , MCPH1 0 1213 6376048 J) Splenectomy chr9: 130092787- 1.03E−6 rs7026795 A 0.425 0.1802 3.363 2 C9orf119 1698 1698 130095025 chr3: 72044083- 1.11E−6 rs7648163 C 0.55 0.2748 3.226 2 AK097190 115138 115138 72052222 chr1: 73821234- 2.23E−6 rs4606267 G 0.325 0.1194 3.552 3 BC041341 244086 244086 73888127 chr9: 70762083- 2.46E−6 rs2993008 T 0.225 0.06306 4.313 2 AK057188, PIP5K1B 0 0 70815910 chr8: 137069094- 3.13E−6 rs6985828 C 0.0625 0.002252 29.53 2 KHDRBS3 340064 340064 137070609 chr15: 66415767- 3.78E−6 rs6494736 C 0.175 0.04054 5.02 2 ITGA11 0 0 66419741 chr7: 141314419- 2.60E−5 rs11761774 A 0.475 0.2455 2.781 2 TAS2R38 4247 4247 141314653 K) Cytopenias chr12: 3711033- 4.28E−6 rs241964 C 0.6111 0.3 3.667 6 C12orf4, C12orf5, CCND2, 0 0 4573405 DYRK4, EFCAB4B, FGF23, FGF6, PARP11, RAD51AP1 chr8: 2721609- 3.52E−5 rs341672 C 0.2593 0.0812 3.961 2 CSMD1, KIAA1890 59615 59615 2723174 chr3: 2628577- 0.000337 rs1020997 G 0.2407 0.4979 0.3198 2 CNTN4 0 40335 2629597 chr2: 1783619- 0.000426 rs6548056 G 0.1852 0.434 0.2963 2 MYT1L 0 514 1783961 chr11: 2240035- 0.000533 rs17659078 A 0.4444 0.2286 2.699 2 ASCL2 5138 5138 2241166 chr10: 1236883- 0.000603 rs10794730 T 0.4815 0.2596 2.649 2 ADARB2 0 583 1237022 chr9: 1588885- 0.000866 rs1923928 T 0.1852 0.4191 0.315 2 SMARCA2 409607 409607 1595735 chr1: 4222578- 0.000884 rs966321 C 0.2593 0.4979 0.353 2 AX748168 146394 146394 4225577 L) organ specific autoimmunity (OSAI) chr8: 101728102- 6.89E−8 rs7815950 G 0.2133 0.05615 4.559 2 SNX31 0 2334 101728344 chrX: 39933689- 5.18E−7 rs2948491 A 0.18 0.04545 4.61 4 BCOR 12163 12163 39956544 chr3: 109180117- 6.27E−6 rs709477 A 0.2733 0.4893 0.3926 2 BC101231 50156 50156 109186346 chr1: 37438130- 6.96E−6 rs6426015 A 0.2667 0.1096 2.953 3 GRIK3 165699 165699 37446852 chr15: 84701881- 1.19E−5 rs1431234 C 0.3 0.5108 0.4105 2 AGBL1 0 20894 84720707 M) Low IgM (<50 mg/dL) chr1: 25735669- 6.02E−8 rs2065970 G 0.06553 0.2411 4.529 5 LDLRAP1 0 0 25753653 chr4: 59393803- 1.77E−5 rs2899130 A 0.2257 0.4286 2.573 2 BC034799 1366894 1366894 59398745 chr5: 76127981- 2.17E−5 rs615986 T 0.4927 0.2679 0.3767 2 F2RL1 21647 21647 76128963 N) Low IgA (<10 mg/dL) chrX: 30733771- 1.44E−7 rs11095197 T 0.1469 0.3431 3.034 2 MAP3K7IP3 18876 18876 30736604 chrX: 145696682- 3.48E−6 rs6626815 C 0.5656 0.3578 0.4279 5 CXorf1 977620 977620 145751200 chr15: 47024768- 6.75E−6 rs17469978 C 0.2313 0.4167 2.375 2 SHC4 0 16198 47025722 chrX: 32356174- 1.25E−5 rs699457 G 0.2656 0.1078 0.3342 2 DMD 0 0 32388539 chr1: 3915418- 2.69E−5 rs10737395 A 0.1 0.2353 2.769 2 AK124708 236 236 3917931 chr13: 101257602- 3.22E−5 rs1322702 A 0.04375 0.1471 3.768 2 FGF14 0 60734 101258342 O) Low B cells (CD19+ cells <1%) chr8: 13834782- 7.62E−8 rs2682665 C 0.1818 0.01394 15.71 3 SGCZ 149368 149368 13842376 chr7: 107306707- 2.16E−7 rs9690688 A 0.3636 0.06375 8.393 2 DLD 4709 4709 107314113 chr9: 33103066- 1.40E−6 rs12379501 T 0.3182 0.05578 7.9 2 B4GALT1 0 0 33103970 chr13: 37798435- 2.25E−6 rs4943583 G 0.2273 0.02988 9.549 7 UFM1 0 0 37846851 P) Young age of symptom onset (<10 yrs) chrX: 152295068- 9.84E−8 rs5987017 A 0.2143 0.04773 5.442 4 ZNF275 24829 24829 152303019 chrX: 37803525- 1.54E−7 rs5918500 C 0.4878 0.2123 3.533 3 SYTL5 0 0 37832251 chr19: 7505735- 2.31E−6 rs604959 C 0.4762 0.2273 3.091 2 PNPLA6 0 0 7508421

The CNV association analysis also uncovered novel genes associated with either the immunopathogenesis of antibody deficiency or the development of specific complications of CVIDs. In fact, 84 CNV deletions and 98 duplications were identified in one or more CVID patients but were not found in any controls. Most were intra-exonic and thus likely to exert impact. Some of the genes potentially affected by the identified CNV were also discovered in the GWAS part of this study. Many others have direct or potential relevance to the immune system and many were unique to individual patients, thus underscoring the great mechanistic diversity that is likely to underlie this collection of disorders, also reflected in the variability in clinical presentation and disease natural history¹. Among those, we noted a highly significant number of subjects with duplications in ORC4L, a gene previously associated with B-Cell lymphoproliferative disorders³³. This gene is essential for initiation of DNA replication, and potentially in rapidly proliferating immune cells.

We then performed a SNP genotype association to the particular features of CVID to define potential common mechanistic threads amongst the specific clinical and immumological variants to enable the prediction of CVID clinical phenotypes. Significant individual associations were made with all CVID variables studied (Table 8) including cancer (1), lymphoma (5), lymphadenopathy (3), NRH (3), LIP (4), bronchiectasis (1), granulomatous disease (0), enteropathy (1), malabsorption (2), splenomegaly (0), cytopenias (0), autoimmunity (2), low IgM (1), low IgA (1), low B cells (2), and early age of onset (2). In this regard, PFTK1 is a member of the CDC2-related protein kinase family found constitutively expressed at high levels in B cell lymphomas³⁴, and also found associated with lymphoma in the studied subjects with this complication. Interestingly, HAVCR1, allele frequency also found enriched in the same subjects, plays a role in Th cell development and the regulation of asthma and allergic diseases³⁵. FGF14, associated with LIP and low IgA, is a member of the fibroblast growth factor (FGF) family, which has crucial roles in embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion³⁶. SNX31, associated with organ-specific autoimmunity, is a sorting nexin which may be involved in protein trafficking; SNX family proteins are subunits required for CD28 mediated T cell costimulation³⁷. LDLRAP1, associated with lower serum IgM, encodes a cytosolic protein which contains a phosphotyrosine binding (PTD) domain which interacts with the cytoplasmic tail of the LDL receptor, and is associated with hypercholesterolaemia. In addition to the potential of these findings to provide mechanistic insight into how these subphenotypes arise, they may also allow for the prediction of associated comorbidity at the time of diagnosis. This has the potential to greatly improve the clinical management of the CVIDs.

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Example 2 Prediction of CVID Status and Development of Complications Based on a Targeted SNP Panels

A Support Vector Machine (SVM) is a decision-based prediction algorithm which can classify data into several groups. SVM is based on training data mapped to a higher dimensional space and separated by a plane defining the two classes of data, followed by a testing stage where status is predicted based on the model and compared to known status to test the accuracy (V. Vapnik. The Nature of Statistical Learning Theory. Springer-Verlag, New York, NY, 1995). The implementation of SVM used is LIBSVM software (Chih-Chung Chang and Chih-Jen Lin, LIBSVM: a library for support vector machines, 2001. Software available at http://www.csie.ntu.du.tw/˜cjlin/libsvm; C. W. Hsu, C. C. Chang, C. J. Lin. A practical guide to support vector classification, 2003). Genome-wide SNP microarrays provide 610,000 genotypes which tag the genome in an unbiased way without need for prior hypothesis concerning candidate genes. Classical genome-wide association methods evaluate single SNP significance for difference in allele frequency. Although successful, the reality that many common complex diseases are polygenic in etiology with epistasis of multiple gene combinations makes an integrative model utilizing more of the data content more incisive. The application of SVM to genome-wide SNP data provides a robust framework for clinical disease prediction to evaluate risk and enact preventative measures, as we have demonstrated previously for type 1 diabetes (Wei Z, et al., From disease association to risk assessment: an optimistic view from genome-wide association studies on type 1 diabetes. PLoS Genet. 2009 October; 5(10):e1000678. Epub 2009 Oct. 9).

There are two components to the data: the target value which is the classification of the subject and the corresponding attributes which are more granular characteristics. In the application to CVID, we sought to predict target Values (Class Labels): CVID (yes/no), Bronchiectasis (yes/no), and OSAI (yes/no). This prediction is based on the attributes (features or observed variables): Genotypes for the top 1,000 SNPs previously identified from case:control genome-wide association (top 0.16% of original data set and all P<0.0015).

Phenotype labels were scaled to +1 for affected and −1 for unaffected. Genotype data is scaled with AA=0, AB=0.5 and BB=1. Data is mapped to high dimensional space by the LIBSVM algorithm to differentiate populations. Iterations of a coarse grid are followed by finer grids to avoid excessive time expenditure of complete grid search. The SVM is based principally on two variables: cost C (the penalty parameter of SVMs) and γ=1/number of variables. The SVM type in our application was C-SVM classification which runs through iterations of minimizing error functions while maintaining a hyperplane with maximum margin to avoid overfitting (FIG. 6). The kernal function maps attribute data into a higher dimensional space to improve distinguishing characteristics. The kernel type in this application was a radial basis function (RBF) which is based on distance from an origin. Once the SVM model is established through training and validated for accuracy with an independent dataset in testing, additional data can be mapped and prediction made based on the model.

The Support vector machine (SVM) approach is a machine learning approach that can map combinations of genotypes in high dimensional space. The top 1000 significant SNPs from the discovery and replication case:control genotype association studies we used as observed characteristics to form a designation decision between CVID (common variable immunodeficiency) case and an individual not affected by CVID (Table 14). The dataset available was separated into a training set of 179 cases and 1,917 controls which the SVM model is based on. In this training set, the case/control label and genotype attributes guide model formation. In the testing set of independent 109 cases and 1,114 controls, only the genotype attributes are supplied to the model established during training which then distinguishes cases or controls based on the genotype profile. The simple diagnostic yes or no question is: based on this person's genotypes do they have CVID? The prediction accuracy turned out well: training data: 98.7% and testing data: 90.5% (FIG. 7).

Similarly, the top 1000 significant SNPs for Bronchiectasis (Table 15) and OSAI (organ specific autoimmunity) (Table 16) as input to the SVM. Training: 107 no Bronchiectasis and 59 Bronchiectasis. Testing: 77 no Bronchiectasis and 19 Bronchiectasis. The model had 100% accuracy in the training stage and 97.9% accuracy in testing stage (FIG. 8). Evaluating our cohort for OSAI yielded Training: 117 no OSAI and 49 OSAI. Testing: 70 no OSAI and 26 OSAI. The model had 100% accuracy in the training stage and 99.0% accuracy in testing stage (FIG. 9).

To demonstrate poor model accuracy based on the same SVM methods, we ran the training stage with randomized testing data. The Bronchiectasis model applied to random affected labels and random genotypes resulted in a prediction accuracy of 27.1%. The Bronchiectasis model applied to random affected labels and correct genotypes resulted in a prediction accuracy of 77.1%. The OSAI model applied to random affected labels and correct genotypes resulted in a prediction accuracy of 67.7%. Given these markedly poorer accuracy results from randomized data compared to our observed data bolsters confidence that our high success rate is less likely due to a confounding factor, bias, or error.

This study represents the first genome-wide population based study of CVID. The use of the relatively large cohorts assembled here was essential, both to discover and to confirm the findings and demonstrates the potential of genome-wide association in complicated polygenic rare diseases. This type of unbiased study has discovered many novel targets that may underlie the development of CVID and provide clues to the pathogenesis of the heterogeneous clinical complications and subtypes of CVID, providing a solid foundation for further studies to understand the mechanism, interplay, and clinical manifestations of CVID, with the immediate potential of improving clinical management. Finally the great diversity identified with regards to unique CNV substantiates the hypothesis that CVIDs are a collection of diverse mechanisms leading to complex phenotypes.

TABLE 14 SNP Panel of 1994 SNPs for classifying CVID case versus individual without CVID rs3117426 rs2239914 rs9382495 rs139234 rs11132547 rs1391772 rs2107202 rs3763338 rs649647 rs7677584 rs2523535 rs366178 rs4269167 rs17631106 rs11135816 rs1400438 rs2112040 rs3763822 rs6504204 rs7690236 rs3130931 rs17304375 rs3130933 rs13019329 rs11138566 rs1411439 rs2113616 rs3766566 rs6505069 rs7716554 rs200968 rs3129871 rs9295768 rs7085343 rs11143520 rs1419183 rs2121289 rs3772054 rs6510040 rs7761966 rs879882 rs1634719 rs967005 rs12270763 rs11157606 rs1421240 rs2137512 rs3775574 rs6516091 rs7762279 rs3130564 rs4654849 rs2395174 rs12447026 rs11180576 rs1422122 rs214833 rs3781216 rs6538408 rs7772160 rs7767008 rs2400955 rs2931060 rs10940184 rs11183395 rs1438935 rs2153875 rs3788111 rs6549225 rs7774197 rs720831 rs6581986 rs10000770 rs139240 rs11206396 rs1443365 rs215393 rs3788317 rs6555424 rs7774567 rs4713208 rs2429485 rs10020322 rs3802888 rs11237730 rs1444467 rs2159318 rs3796504 rs656070 rs7781284 rs9266689 rs3129975 rs12636521 rs17790790 rs11247770 rs1445358 rs2168963 rs3801332 rs6577933 rs7789182 rs13194504 rs17099394 rs139136 rs4871793 rs1157548 rs1447398 rs217180 rs3804350 rs6583700 rs7808907 rs6926142 rs2395173 rs9380006 rs2279529 rs11669334 rs1452077 rs2175835 rs3810925 rs6586116 rs7815122 rs4872262 rs12495023 rs10476080 rs2902858 rs11698275 rs1461713 rs220309 rs3814585 rs6590124 rs7822769 rs12680982 rs10259703 rs358994 rs4720301 rs11717880 rs1466633 rs2213215 rs3818409 rs6656611 rs7834603 rs6456785 rs2505323 rs12573587 rs17372123 rs11786911 rs1477246 rs2236861 rs3847985 rs6657001 rs783756 rs2517532 rs2505327 rs139135 rs6679430 rs11819553 rs1481150 rs2237236 rs3863380 rs6688151 rs7838893 rs2156875 rs2484173 rs3095340 rs6485702 rs1182865 rs1497431 rs2242660 rs3913305 rs6688807 rs7857243 rs3130501 rs3132685 rs11087123 rs3134792 rs11848008 rs149990 rs2256726 rs3935608 rs6694182 rs7857474 rs422717 rs2523608 rs9266440 rs1377611 rs11862051 rs1523138 rs2262808 rs4082339 rs6727285 rs7867936 rs393034 rs3094122 rs2718419 rs9380120 rs11873322 rs1525745 rs2277210 rs4082846 rs6737948 rs7871753 rs3117326 rs8056528 rs1194849 rs6799262 rs11906 rs1544488 rs2292263 rs4146035 rs6743415 rs7887141 rs3129882 rs879484 rs7718291 rs12495026 rs11915523 rs1545692 rs2292307 rs4147525 rs6759577 rs7904468 rs3130837 rs2517549 rs12458578 rs2523989 rs11924324 rs1566579 rs2295330 rs420095 rs6764451 rs791033 rs8321 rs10734912 rs6426636 rs7945342 rs11933720 rs1569583 rs2299395 rs4236541 rs6794896 rs7928480 rs6571989 rs2523987 rs469709 rs950210 rs1194491 rs1571272 rs2306810 rs4238992 rs6807774 rs7937011 rs9261290 rs3130932 rs9329172 rs1406977 rs11959994 rs1574342 rs2343468 rs4320356 rs6865181 rs7939415 rs858985 rs2210313 rs13104213 rs720465 rs11963652 rs1582761 rs2358483 rs4324798 rs6877998 rs7964472 rs1606234 rs3135338 rs1876518 rs11870286 rs11977312 rs1592593 rs2373146 rs4353961 rs6886088 rs7970177 rs3129791 rs3770266 rs2572690 rs9257802 rs11998428 rs16181 rs2389789 rs4384353 rs6903608 rs8015024 rs12924882 rs149900 rs2191388 rs2517403 rs12001157 rs1619092 rs2406233 rs4409432 rs6904596 rs8015785 rs4264554 rs6457327 rs3094127 rs7103569 rs12047230 rs1629896 rs2425628 rs4486000 rs6910095 rs8020688 rs17322265 rs4871402 rs386843 rs12374025 rs12049377 rs1652500 rs2427399 rs4497887 rs6917366 rs8038569 rs1453666 rs2517448 rs17099425 rs10746192 rs12060746 rs1654774 rs2448396 rs4512403 rs6917419 rs8050560 rs7087554 rs2902982 rs310319 rs10254238 rs120960 rs1658102 rs2455826 rs4525114 rs6921388 rs8055387 rs853676 rs493965 rs2733393 rs139235 rs1209633 rs16891725 rs2473700 rs4531492 rs6923139 rs8057776 rs3771781 rs80303 rs1539120 rs3749530 rs12146261 rs16899857 rs2492460 rs454127 rs6928830 rs8061733 rs203888 rs9295730 rs2844494 rs2844635 rs12147922 rs16908361 rs2497295 rs4541737 rs6933251 rs8068923 rs3094551 rs200969 rs133826 rs1517927 rs12148329 rs16949286 rs2514861 rs4578658 rs6938076 rs8077733 rs1634718 rs1028308 rs9344757 rs17251715 rs12149564 rs16956295 rs2517646 rs4596286 rs6939576 rs8081687 rs3094550 rs3130350 rs1214751 rs9313540 rs12188351 rs16957467 rs2523454 rs4617771 rs6947841 rs8100750 rs6127923 rs2254556 rs2130904 rs4354950 rs12190473 rs16959820 rs2535319 rs464904 rs6959814 rs8105356 rs1064191 rs17293544 rs2730306 rs6785581 rs12201890 rs16971464 rs2554675 rs4671614 rs6960076 rs8109860 rs2517452 rs393990 rs12145634 rs2454873 rs12204145 rs17043526 rs2555575 rs4689343 rs6961642 rs8138344 rs9257809 rs16851846 rs4350445 rs5009448 rs12238437 rs17052298 rs2560623 rs4692098 rs6972083 rs821107 rs895710 rs1533476 rs606448 rs10018912 rs12258095 rs17090892 rs258229 rs4695718 rs697614 rs835361 rs149951 rs940052 rs7617297 rs10028187 rs12261686 rs17100060 rs2589661 rs4695791 rs6981398 rs835365 rs6043091 rs12924838 rs10255854 rs10058261 rs12275853 rs17100227 rs2598464 rs4712728 rs6981439 rs835378 rs3749971 rs10746490 rs12577638 rs10100444 rs12276737 rs17199261 rs2629228 rs4712955 rs6993922 rs835435 rs12153912 rs12445992 rs2442749 rs10101117 rs12291520 rs1733795 rs2636767 rs4712984 rs6998016 rs838893 rs3094663 rs9468276 rs10916754 rs1013696 rs1233579 rs17398230 rs26685 rs4737520 rs7005380 rs855974 rs175597 rs12093075 rs7783758 rs1013849 rs1233708 rs17400329 rs2695636 rs4754214 rs7011777 rs872111 rs200953 rs2517552 rs7463896 rs1014028 rs1235162 rs17422797 rs2708594 rs4758294 rs7037024 rs879921 rs9257805 rs3132538 rs11780509 rs10148024 rs12405434 rs17431811 rs2708603 rs4763780 rs7041937 rs886403 rs1545523 rs3130361 rs2844729 rs1015502 rs12432589 rs17434960 rs2716456 rs4764038 rs7096350 rs886424 rs400404 rs4894920 rs10501763 rs1018393 rs12444655 rs17436819 rs2745061 rs4764589 rs7098890 rs888932 rs200951 rs910064 rs719927 rs10189525 rs12452324 rs17476501 rs2746150 rs4765477 rs7100025 rs905780 rs10494349 rs12491592 rs10517239 rs10199875 rs1247571 rs17595027 rs2755223 rs4766329 rs7108362 rs9262143 rs9950880 rs1466065 rs7657149 rs10212407 rs12563394 rs17616999 rs2765283 rs4768814 rs7109698 rs9267947 rs17803433 rs11784192 rs9921767 rs10224415 rs12571401 rs17637266 rs2777877 rs4772030 rs7110148 rs9271366 rs12712943 rs2055918 rs6679380 rs10240549 rs12574668 rs17641748 rs2791333 rs4787348 rs7122039 rs9275602 rs3130544 rs2306029 rs2516398 rs10255489 rs12577504 rs17663626 rs2808416 rs4814335 rs7130141 rs9295794 rs17749927 rs1609942 rs11081638 rs1036165 rs1264514 rs17699790 rs280986 rs4823340 rs7130640 rs9302358 rs13062596 rs3091661 rs7193689 rs1039128 rs12665573 rs17735101 rs2813299 rs4833753 rs7141603 rs9357078 rs9444253 rs1468042 rs3856154 rs1041981 rs12679478 rs17742080 rs2815067 rs484870 rs7149428 rs9366694 rs7514144 rs16867404 rs17016917 rs10428921 rs12680797 rs17768474 rs2822670 rs4849333 rs717655 rs9373537 rs13194781 rs2905722 rs538186 rs10464032 rs12726611 rs17798693 rs2823594 rs4862110 rs7193708 rs9379858 rs3130827 rs1196493 rs3767996 rs10484399 rs12735814 rs181027 rs2823691 rs4863305 rs7216116 rs9380064 rs7750641 rs7775818 rs7707008 rs10484560 rs12744234 rs181046 rs2833979 rs4899866 rs7217235 rs9393796 rs506659 rs3095329 rs10946966 rs10486023 rs12787569 rs1810636 rs2834034 rs4905713 rs7224103 rs9410486 rs9348772 rs1396400 rs10994852 rs10491190 rs12801193 rs1847039 rs2836293 rs4943552 rs7233676 rs9436188 rs9257696 rs17047183 rs9380007 rs10491964 rs12807746 rs1853156 rs2859365 rs4948286 rs726532 rs944963 rs7972182 rs359030 rs3099844 rs10500817 rs12883308 rs1858339 rs287482 rs4957798 rs7267722 rs9468209 rs6940139 rs1012411 rs2217046 rs10513315 rs12892005 rs1863050 rs2876038 rs4968363 rs727708 rs9468213 rs10951566 rs4320355 rs200997 rs10514840 rs12898202 rs1874655 rs2913277 rs497322 rs7305297 rs9468413 rs853679 rs1470539 rs3094061 rs10517209 rs12908686 rs1879689 rs2953807 rs497879 rs736884 rs9472202 rs3132631 rs2125639 rs2833297 rs10517365 rs12969251 rs1884123 rs2959187 rs529655 rs737448 rs9474687 rs3132630 rs7168491 rs2516675 rs1059260 rs12971478 rs1922407 rs2974101 rs534451 rs740791 rs947950 rs287355 rs9322400 rs6812482 rs1073320 rs13077924 rs1932422 rs3019885 rs535842 rs740854 rs9529418 rs12102284 rs9384054 rs2844670 rs10736156 rs13093086 rs1935135 rs3095314 rs545833 rs744280 rs9574659 rs11028465 rs258873 rs2473520 rs10741559 rs13095274 rs1951805 rs3105403 rs547136 rs7455060 rs957566 rs9380069 rs6894136 rs473925 rs10746190 rs13144404 rs1952790 rs3108396 rs553219 rs7463839 rs9578469 rs7115116 rs9884830 rs10238440 rs10757394 rs13164993 rs1955490 rs3116817 rs556723 rs7503953 rs958731 rs409783 rs9382494 rs6911628 rs10760260 rs1316537 rs1955522 rs3117143 rs558560 rs751717 rs9598293 rs2395182 rs693196 rs3094188 rs10795445 rs13185914 rs1978454 rs3128982 rs5758991 rs752479 rs9608247 rs4768959 rs16972292 rs734585 rs10804253 rs13195509 rs1982672 rs3130380 rs6013355 rs7543122 rs9635993 rs203890 rs1438544 rs4903707 rs10828096 rs13197574 rs1994667 rs3130817 rs6017996 rs7545388 rs968770 rs6580783 rs11178549 rs133807 rs10843894 rs13198474 rs1997960 rs3130893 rs603836 rs7565134 rs969672 rs1264551 rs10916755 rs2168801 rs10845073 rs13207908 rs2014308 rs3131093 rs6049804 rs7584650 rs9767240 rs2048507 rs12922855 rs12137004 rs10902485 rs13208164 rs2027858 rs3132486 rs6083557 rs7584862 rs982234 rs12532319 rs1196488 rs9295131 rs10924845 rs13211507 rs203877 rs3132580 rs6084833 rs7588316 rs9837352 rs1264547 rs301396 rs2143245 rs10935431 rs13240982 rs2039290 rs3132610 rs618746 rs7617594 rs9842506 rs4558863 rs258862 rs12623733 rs10946940 rs13274015 rs2042280 rs3134603 rs621310 rs7623321 rs9846423 rs3094054 rs573118 rs847067 rs10955450 rs13297297 rs2060321 rs3134954 rs637384 rs7628034 rs9846545 rs287351 rs17273893 rs8069430 rs11026531 rs13297471 rs2066363 rs328294 rs638324 rs7630157 rs9850579 rs3770267 rs12417962 rs3823055 rs11028548 rs1332226 rs2067869 rs338479 rs6456834 rs7634908 rs9879707 rs974332 rs3781703 rs2833293 rs11032297 rs1344723 rs2071591 rs347965 rs6469858 rs7636581 rs9882269 rs11207520 rs12637203 rs6504930 rs11071519 rs13448 rs2076305 rs371915 rs6473735 rs764971 rs9923061 rs2048508 rs1503920 rs1364290 rs11078454 rs1349951 rs2076346 rs3755458 rs6482358 rs7651055 rs9934109 rs12193434 rs869340 rs2047456 rs11084476 rs1366963 rs2076537 rs3759864 rs6482573 rs7654623 rs997448 rs2285415 rs569078 rs9303355 rs11123677 rs1387815 rs2076624 rs3761093 rs6485690 rs766127 rs999900 rs12086296 rs1953858 rs7661151 rs9938215 rs11588952 rs13252630 rs1894090 rs2985902 rs58901 rs7576475 rs12095205 rs6864519 rs4257189 rs7979863 rs11591167 rs1326415 rs1898462 rs3007290 rs596167 rs7580465 rs1886740 rs11144516 rs12507717 rs1000408 rs1159372 rs13275988 rs1923105 rs302113 rs6008581 rs7583467 rs875688 rs6470066 rs34687 rs10014832 rs11617865 rs13290774 rs1925690 rs303545 rs6011734 rs7591430 rs230177 rs7143400 rs2594897 rs10021608 rs11619258 rs13336037 rs1932169 rs30425 rs6024661 rs761021 rs2652130 rs12155344 rs1476994 rs10022535 rs11635501 rs1336892 rs1944888 rs3095089 rs6024723 rs7634304 rs6728658 rs486238 rs13263545 rs10032135 rs11637538 rs1338588 rs1952098 rs3133250 rs6028945 rs7669997 rs10833106 rs316013 rs6982249 rs1003991 rs11654639 rs1342602 rs1956521 rs316003 rs6056595 rs7673654 rs230178 rs10806731 rs2769004 rs10040906 rs11670621 rs1353681 rs1975920 rs316780 rs6062957 rs7684327 rs6489056 rs598908 rs282416 rs10053502 rs11675971 rs1365016 rs1995598 rs328144 rs6064315 rs7711139 rs2435962 rs2256141 rs2585197 rs10065046 rs11683424 rs136631 rs1995863 rs330792 rs6065409 rs7728954 rs1391049 rs10484893 rs7140766 rs10070799 rs11720980 rs1374460 rs2001087 rs353826 rs6069541 rs7731260 rs9380602 rs7798161 rs11157866 rs10090889 rs11727494 rs1385340 rs2027363 rs35402 rs6074798 rs7743003 rs3734078 rs10440376 rs40507 rs10092250 rs11728925 rs139394 rs2038359 rs3739464 rs6113074 rs7752724 rs10744242 rs1333217 rs4624543 rs10114302 rs11735072 rs1401014 rs2043388 rs3743466 rs6123013 rs7754710 rs2447753 rs10899269 rs709012 rs10163150 rs11735242 rs1407179 rs2045058 rs3751221 rs6124082 rs7772299 rs11058480 rs2059058 rs12537855 rs10188058 rs11749633 rs1411456 rs2050198 rs3774285 rs634265 rs7773199 rs230174 rs7962622 rs580333 rs1020679 rs11760907 rs1432237 rs2054017 rs3777102 rs6469525 rs7784465 rs6661744 rs3783870 rs904962 rs10242195 rs11770757 rs1447826 rs2068914 rs3781408 rs6473241 rs7786745 rs10753109 rs11982298 rs10225065 rs10243212 rs11776451 rs1455311 rs2073917 rs3803004 rs6491316 rs7800935 rs10934667 rs13253691 rs923963 rs10256012 rs11784473 rs1462559 rs2076370 rs3808528 rs6508244 rs7805356 rs10222858 rs975317 rs4675438 rs10265223 rs11862457 rs1464728 rs209445 rs3813218 rs6566546 rs7837249 rs8008081 rs11622145 rs34689 rs10275033 rs11867922 rs1469587 rs2108294 rs3817614 rs6568006 rs7867090 rs34677 rs9811068 rs7154632 rs1027604 rs11905518 rs1492178 rs2138244 rs3845732 rs6571692 rs7894314 rs11058562 rs1007289 rs2019214 rs10276544 rs11914777 rs1502795 rs2161393 rs3846911 rs6572412 rs790838 rs1480548 rs1373928 rs7034698 rs1036819 rs1192380 rs1502815 rs2165419 rs3850590 rs6578882 rs7911956 rs10742380 rs1373930 rs11237688 rs1039326 rs11933412 rs1503854 rs2165440 rs3857404 rs6580288 rs7926934 rs10752798 rs1461780 rs2830706 rs10407008 rs11947802 rs1510909 rs2174804 rs3858336 rs6589849 rs7929621 rs2110328 rs4721679 rs6766607 rs10417646 rs11955226 rs1519982 rs2181139 rs3864883 rs6594352 rs7938 rs9364554 rs6676552 rs1007764 rs1043595 rs1203181 rs1528461 rs2193976 rs38964 rs6600320 rs7941806 rs1384862 rs3887233 rs3125056 rs1047033 rs12037664 rs1535060 rs2194232 rs3909722 rs6659944 rs7960169 rs7517989 rs10850917 rs2685513 rs10481751 rs12039124 rs1539057 rs2195101 rs3912477 rs666432 rs7976970 rs10799220 rs10898550 rs1029508 rs10483284 rs12039194 rs1546120 rs2204506 rs3923626 rs6672510 rs7981535 rs11684509 rs9301487 rs11167794 rs10483345 rs12102589 rs1549343 rs2221705 rs4012731 rs6689095 rs7986486 rs7679866 rs17124780 rs1440954 rs10483749 rs12113865 rs1555649 rs2236479 rs404220 rs6692418 rs8006182 rs10003811 rs3755665 rs540697 rs10483752 rs1211864 rs1556190 rs224632 rs4078315 rs6700346 rs8014539 rs1593398 rs1444756 rs10498440 rs10484710 rs12119869 rs1559101 rs225132 rs4237471 rs6705017 rs8021964 rs12401659 rs6809208 rs4917727 rs10485722 rs12124645 rs1570677 rs2255313 rs4239638 rs6719977 rs8024016 rs10933421 rs2045838 rs33352 rs10487264 rs12157917 rs157198 rs2271359 rs4239875 rs6723590 rs8027714 rs17284917 rs12703848 rs13169010 rs10490741 rs12187771 rs1572312 rs2275979 rs4246703 rs6726265 rs8057939 rs997131 rs10913056 rs10765174 rs10493695 rs12194954 rs1617406 rs2278404 rs4247190 rs6741951 rs8076084 rs11723364 rs17485462 rs1422780 rs10496080 rs12197035 rs16837801 rs2279664 rs4251739 rs6747421 rs8079249 rs9997989 rs7754676 rs16840921 rs10497071 rs12206076 rs16842293 rs2279986 rs432959 rs6756097 rs808338 rs3746406 rs649611 rs10226333 rs10497422 rs12210297 rs16870964 rs2285487 rs4359096 rs6762119 rs8086871 rs11721736 rs2289709 rs4270027 rs10507882 rs12292437 rs1687503 rs2287904 rs4360077 rs6763408 rs8100291 rs17633196 rs1373931 rs2816316 rs10508006 rs12298502 rs16903944 rs2287906 rs4376903 rs6790285 rs8107145 rs15612 rs17347590 rs12536790 rs10508704 rs12345213 rs16942077 rs2291220 rs4385337 rs6805004 rs8118662 rs6430676 rs2116280 rs7818198 rs10514583 rs12358866 rs16945809 rs2292645 rs4448310 rs6824270 rs813328 rs6775178 rs1471403 rs866279 rs10514908 rs12360927 rs16957064 rs2300520 rs4482854 rs6832174 rs816812 rs2245199 rs9873621 rs659450 rs10516548 rs12458088 rs16959254 rs2302107 rs4523256 rs6833298 rs84823 rs16827675 rs7044480 rs12903340 rs10518315 rs12486791 rs16973160 rs2303663 rs4542999 rs6853448 rs876458 rs2607374 rs1005051 rs11979495 rs10519160 rs12489686 rs16976984 rs2328014 rs4557653 rs6860662 rs878357 rs2093045 rs7863610 rs6732762 rs10519460 rs12493932 rs17019212 rs2332074 rs4572252 rs6864345 rs885157 rs184968 rs2809285 rs6717822 rs10521955 rs12496156 rs1704589 rs233251 rs4598609 rs6883259 rs888186 rs17551608 rs2594909 rs1510868 rs1052653 rs12511504 rs17047071 rs2344553 rs4600036 rs6883877 rs892458 rs2396327 rs6771029 rs4242363 rs1064524 rs12511535 rs17089390 rs2348553 rs4611118 rs6890880 rs905067 rs6426408 rs10799632 rs10483709 rs10734418 rs1253690 rs170974 rs2354040 rs4623688 rs6892994 rs914405 rs2236561 rs10915494 rs12261638 rs10738758 rs12541121 rs17106642 rs2365491 rs4636656 rs6936594 rs9320043 rs607138 rs2984920 rs1440950 rs10764760 rs12549864 rs17114086 rs2373974 rs4662336 rs6982126 rs933235 rs11086147 rs643556 rs4072280 rs10783289 rs12565883 rs17125944 rs2394527 rs4664723 rs6995299 rs9346816 rs16980240 rs913834 rs13275678 rs10793139 rs12566065 rs17128651 rs2397355 rs4700281 rs7000759 rs9365900 rs17149499 rs10409727 rs2805533 rs10801172 rs12599653 rs17134117 rs2407999 rs4703363 rs7002144 rs9366027 rs6957548 rs656635 rs2476779 rs10801415 rs12601054 rs17147479 rs2408347 rs4709877 rs7014566 rs9396682 rs11196077 rs2288898 rs12255933 rs108042 rs12602618 rs17164435 rs2442579 rs4721879 rs7016252 rs9431471 rs4468370 rs2288897 rs479772 rs10818014 rs12656332 rs17172860 rs245891 rs4732639 rs7029119 rs944223 rs2177627 rs13127257 rs1040049 rs10841511 rs12669415 rs17176792 rs2462692 rs4736424 rs704010 rs9445788 rs630760 rs2902990 rs4233164 rs10847189 rs12676720 rs17184300 rs247325 rs4760176 rs704265 rs9453898 rs1323292 rs6019266 rs1111366 rs10847335 rs12698419 rs17193036 rs2513938 rs4766885 rs7043995 rs9460864 rs610490 rs7044607 rs2971927 rs10875883 rs12718513 rs17208897 rs2520570 rs4771059 rs706293 rs9535048 rs7613062 rs7295136 rs504093 rs10894155 rs12727642 rs17214357 rs2553226 rs4771063 rs7114098 rs9536654 rs9290973 rs13262033 rs933153 rs10921466 rs12733466 rs17224695 rs2553600 rs4778871 rs712414 rs9556934 rs1537479 rs4316158 rs2883367 rs10921627 rs127382 rs17241282 rs260879 rs4785434 rs712457 rs9572312 rs7613679 rs7681088 rs10997263 rs10924303 rs12744534 rs1725132 rs2613179 rs4808652 rs7137203 rs9601157 rs2168659 rs2161299 rs836556 rs10928866 rs12746344 rs17314091 rs2623702 rs4809958 rs7153143 rs9608024 rs6128302 rs3914329 rs7150859 rs10932158 rs12748679 rs17365609 rs2630397 rs4810622 rs7160582 rs9769000 rs10847227 rs2703978 rs4262641 rs10940024 rs12769098 rs17389911 rs26775 rs4856002 rs717163 rs977712 rs474304 rs7107322 rs4146018 rs10944479 rs12781083 rs17402243 rs2691782 rs4858594 rs71815 rs977799 rs6726035 rs2769006 rs11749794 rs10961505 rs12813396 rs1748329 rs269718 rs4859011 rs719428 rs978268 rs3746405 rs2295746 rs17562787 rs10968347 rs12817342 rs17521729 rs270050 rs4859325 rs7250113 rs9784568 rs2702671 rs1353583 rs10822856 rs10984477 rs1281747 rs17548847 rs2703130 rs4861993 rs725579 rs9809600 rs1893306 rs7962880 rs6542736 rs10984490 rs12827322 rs17627259 rs2703960 rs4864553 rs726558 rs9809773 rs315995 rs6714893 rs9844737 rs1100185 rs12882217 rs17654772 rs274646 rs4865815 rs7272481 rs982511 rs12155045 rs7171256 rs1316471 rs11014455 rs12885228 rs1765886 rs2755176 rs4885371 rs728034 rs9835081 rs6128300 rs1003802 rs236410 rs11022509 rs1289501 rs17685418 rs2763419 rs4901523 rs729154 rs9848911 rs11666885 rs13214001 rs473585 rs11033019 rs12982321 rs17692241 rs2802884 rs4915723 rs729648 rs9861236 rs2685515 rs316021 rs2420175 rs11059821 rs12990534 rs17709684 rs2817200 rs4920566 rs731809 rs9874349 rs6748817 rs4972265 rs3886771 rs11128687 rs12996858 rs17726892 rs2821236 rs4921593 rs7321884 rs988158 rs2279351 rs1650817 rs2025633 rs11131055 rs13023918 rs17734487 rs2822850 rs4933679 rs734434 rs988179 rs11059258 rs7265772 rs6012463 rs11143230 rs13035689 rs17761855 rs2825673 rs494469 rs7349465 rs9904523 rs4130446 rs7724262 rs3849699 rs11165460 rs1304065 rs17796406 rs2852424 rs4950989 rs739107 rs9912125 rs7084512 rs251039 rs12188691 rs11186642 rs13081891 rs17817943 rs2877498 rs4976254 rs740096 rs9913538 rs627830 rs2057904 rs9325837 rs11194816 rs13086978 rs17829645 rs2887022 rs4978774 rs7415038 rs9919607 rs2052701 rs12608393 rs2010464 rs11218881 rs13128635 rs178377 rs2904295 rs4981149 rs7428676 rs9966000 rs1475784 rs2475793 rs13394087 rs1124359 rs13132576 rs1790024 rs2914079 rs4984626 rs752278 rs9989335 rs6419277 rs2455308 rs11237690 rs11250154 rs13142179 rs1833755 rs2924264 rs500293 rs7539255 rs6769238 rs1808378 rs2152174 rs1146229 rs13189435 rs1861089 rs2940930 rs536299 rs7544671 rs7306734 rs4521737 rs4947602 rs1152986 rs13203110 rs186493 rs296200 rs542988 rs7553961 rs3125050 rs547491 rs6437079 rs11547160 rs13224666 rs1866714 rs2962270 rs556439 rs7554157 rs2174559 rs1477602 rs7609626 rs1156250 rs13229792 rs1885831 rs2972418 rs568789 rs7555638 rs6661074 rs7792461 rs722938 rs11576361 rs13238853 rs1889356 rs2974617 rs587263 rs7574670

TABLE 15 SNP Panel of 1000 SNPs for classifying CVID case with Bronchiectasis versus CVID case without Bronchiectasis rs3101219 rs1866236 rs9837945 rs7700961 rs6950759 rs7021834 rs6590583 rs8011776 rs6508240 rs5925649 rs4908463 rs1349159 rs13083177 rs297884 rs2191714 rs1536864 rs6590584 rs7174078 rs2120663 rs5925651 rs1725262 rs1448917 rs960353 rs7735038 rs2330055 rs11142904 rs6590694 rs11858397 rs16946189 rs5926027 rs10864368 rs12612313 rs7629105 rs1050975 rs11238193 rs7037278 rs546381 rs17227996 rs1350552 rs5926032 rs2071414 rs6436652 rs6443582 rs1890366 rs296308 rs2378383 rs11054372 rs12439272 rs621834 rs7882511 rs12034719 rs9288624 rs1522262 rs1933650 rs11766378 rs7025226 rs2071163 rs4924613 rs2433396 rs1527808 rs2982380 rs1515928 rs1994855 rs6904644 rs10499751 rs12238088 rs2239176 rs1712426 rs2847593 rs5943997 rs17038468 rs2556097 rs12487104 rs1331489 rs10270164 rs10491791 rs901528 rs3825786 rs7241842 rs12557438 rs1812242 rs1561466 rs4602335 rs1571827 rs13240999 rs7874528 rs10841413 rs12439521 rs16977580 rs16988109 rs4970535 rs13033944 rs4472002 rs9391956 rs12698917 rs17428350 rs2417862 rs7178085 rs1942159 rs12014989 rs12126652 rs830994 rs4234788 rs3812179 rs258702 rs7871887 rs2203494 rs11855605 rs3753067 rs1419850 rs12131682 rs831007 rs7435615 rs3812178 rs2888019 rs10818955 rs11045417 rs2306335 rs11152012 rs5973335 rs272561 rs831011 rs4697075 rs7760489 rs1557785 rs1413294 rs1861710 rs1993865 rs11663936 rs728149 rs1051648 rs6707969 rs2167955 rs966840 rs1921598 rs2114012 rs10492367 rs1026695 rs10516006 rs7064305 rs3444 rs16862458 rs5002502 rs13196069 rs1526491 rs3847303 rs7307357 rs4776191 rs17638216 rs5972346 rs6667593 rs6433615 rs7665747 rs12665403 rs7781914 rs328875 rs2388962 rs1904109 rs7251154 rs2124748 rs2354463 rs13032587 rs12186184 rs1905212 rs2188508 rs7852829 rs10844021 rs17183491 rs10416824 rs228379 rs10493140 rs17362588 rs1377347 rs9476488 rs17166818 rs1385143 rs1666235 rs3809539 rs6508999 rs2180648 rs10888636 rs2290517 rs13116757 rs9358858 rs1015882 rs4979619 rs10743775 rs1865930 rs7254214 rs5917579 rs2806405 rs12465639 rs7349609 rs6904130 rs2158137 rs12236795 rs12319134 rs11858355 rs1594895 rs1800321 rs2764687 rs10204810 rs4527518 rs2524099 rs10275909 rs7020797 rs995342 rs12914140 rs2217672 rs5917584 rs12728521 rs4667046 rs10034992 rs9501626 rs7777145 rs10481656 rs1389134 rs8032153 rs8113456 rs11266207 rs17124275 rs785240 rs1000226 rs3135338 rs3735258 rs7049083 rs1498712 rs11639228 rs6509286 rs5917593 rs4512683 rs12613687 rs2622604 rs2027856 rs234 rs7041855 rs10877894 rs4436753 rs689292 rs5964215 rs11208446 rs6740981 rs1708670 rs2395173 rs17356935 rs1249904 rs11174573 rs4534816 rs3746660 rs2239455 rs12407601 rs10445792 rs583908 rs154978 rs7455060 rs2225067 rs3936640 rs6497041 rs6084506 rs10854983 rs6663109 rs4673837 rs4693223 rs958423 rs10272242 rs10818337 rs3864455 rs11635007 rs998132 rs5919551 rs7522367 rs6739563 rs10049681 rs804829 rs6466735 rs10984619 rs4763120 rs768399 rs945767 rs5919560 rs11208834 rs11687313 rs10516451 rs1109798 rs4726499 rs11790238 rs11175217 rs8024991 rs6139278 rs5936698 rs1415974 rs4672726 rs4833233 rs3807045 rs2001942 rs6478430 rs962415 rs12591805 rs6076598 rs7056340 rs3738168 rs2043769 rs1155135 rs2257082 rs12703419 rs1860665 rs10506554 rs13380379 rs6516091 rs6625489 rs787492 rs13019534 rs1480902 rs941967 rs855740 rs2297454 rs10506762 rs4965678 rs6054459 rs5936708 rs7541725 rs12053340 rs1383518 rs2268718 rs1111467 rs10818649 rs919993 rs7177883 rs2206423 rs1327347 rs6691042 rs1527944 rs13125153 rs4391265 rs1079060 rs10988617 rs2468358 rs9944290 rs2050104 rs5936488 rs617196 rs1527947 rs12503254 rs1377392 rs2952648 rs11103479 rs7300004 rs11640138 rs8120907 rs5936735 rs263463 rs16836294 rs1456266 rs10948750 rs11777063 rs10905076 rs10777720 rs415595 rs2223565 rs6418420 rs263495 rs17450826 rs951850 rs9342394 rs6558946 rs12781427 rs3782518 rs949429 rs1033470 rs5936510 rs12409961 rs12472818 rs4569797 rs701699 rs7816614 rs10752197 rs2293055 rs12918743 rs4813897 rs5980665 rs2295330 rs16858496 rs939689 rs493265 rs1526371 rs7092558 rs11615235 rs7404377 rs6040808 rs5936518 rs11184300 rs12464171 rs6536370 rs9351817 rs2527748 rs7897007 rs4760566 rs238848 rs2009018 rs5936520 rs11184318 rs10933001 rs6858744 rs199623 rs13254568 rs11816696 rs2129663 rs2521477 rs6110058 rs5936795 rs2765283 rs12479209 rs2047633 rs12207816 rs7836269 rs12254001 rs7134809 rs2910848 rs17272940 rs5936796 rs7516108 rs1400795 rs13140600 rs16881643 rs2736020 rs749631 rs7300570 rs8045557 rs6079923 rs5936524 rs401904 rs17371273 rs2706740 rs10485242 rs1563297 rs2793345 rs12422489 rs763763 rs6046129 rs3811372 rs1748383 rs6711657 rs2706727 rs16881894 rs2720611 rs7071467 rs1334958 rs3935714 rs7267722 rs749789 rs7523184 rs12463466 rs2660404 rs10944450 rs4831774 rs7911971 rs9510802 rs257868 rs6049003 rs7879974 rs1986860 rs7355380 rs6841955 rs16881936 rs10503553 rs4256883 rs4771142 rs9926100 rs6121337 rs6615046 rs4233394 rs11685503 rs2082317 rs7754169 rs822249 rs2138566 rs1336628 rs7199018 rs228838 rs12012447 rs4540634 rs2645779 rs11729502 rs2518321 rs822304 rs10822031 rs4771033 rs7188866 rs6126431 rs5958896 rs1986385 rs1705805 rs11725347 rs12212740 rs11203812 rs10996997 rs903777 rs8049176 rs6069746 rs6621735 rs2766085 rs17709863 rs6818908 rs1858234 rs11204097 rs10762038 rs1952994 rs8047279 rs438363 rs5985075 rs12062372 rs356418 rs11733679 rs4371861 rs12545707 rs2894317 rs391933 rs16959476 rs160396 rs5942366 rs10800150 rs6769422 rs10024068 rs6913350 rs7841346 rs890687 rs398655 rs12449237 rs1151621 rs5942534 rs10918281 rs9875884 rs7698980 rs1933755 rs2942202 rs1550023 rs9603418 rs4458015 rs6062292 rs5941436 rs4472738 rs11129323 rs2280664 rs9492657 rs2685320 rs1684543 rs4346095 rs8057941 rs8129104 rs1570995 rs6680341 rs1015856 rs2692596 rs2249821 rs11136019 rs4547043 rs9526255 rs17296283 rs443823 rs6522689 rs10489199 rs9837352 rs1288536 rs2807278 rs7833986 rs2497304 rs4534729 rs13338146 rs2407255 rs638376 rs2205851 rs33454 rs6849285 rs2024692 rs7822429 rs10736156 rs17069883 rs17712938 rs2825809 rs12833867 rs232293 rs3821477 rs11724261 rs9403904 rs4397440 rs10884480 rs9534596 rs11648482 rs2825817 rs6644233 rs1933539 rs971866 rs957792 rs4870376 rs7824497 rs10748948 rs1323537 rs8055798 rs2825819 rs12556735 rs6593937 rs715382 rs1444127 rs9322536 rs924740 rs10749189 rs9535080 rs16955356 rs2826213 rs5910956 rs12061474 rs7647357 rs16892616 rs12174424 rs1545824 rs690870 rs777771 rs2317835 rs236044 rs4373692 rs11579791 rs6810180 rs4869452 rs2770104 rs2128105 rs7073467 rs9598119 rs378138 rs5992088 rs204330 rs10489951 rs6445254 rs6883671 rs1118242 rs7832751 rs3011399 rs2137512 rs1946482 rs2535707 rs203490 rs892975 rs11130981 rs1466011 rs9654570 rs7015186 rs3011408 rs7988149 rs8081951 rs361893 rs16309 rs6426156 rs1283532 rs2910468 rs751873 rs6472384 rs7070793 rs3013580 rs7219019 rs849357 rs201637 rs4653662 rs7373042 rs2910469 rs9456433 rs11778612 rs1898463 rs4884738 rs17762680 rs1297593 rs149908 rs973252 rs13100924 rs1423131 rs4368835 rs17824659 rs11248595 rs7997966 rs7215084 rs1807510 rs2046394 rs2808611 rs6548962 rs2910475 rs10214534 rs6472770 rs11244515 rs9317784 rs7503953 rs738456 rs683335 rs2808614 rs7610023 rs2961838 rs2457571 rs7461233 rs4962566 rs2325194 rs4792143 rs392277 rs546562 rs11122322 rs6548984 rs1862526 rs9365646 rs5017296 rs12098358 rs1446385 rs4393623 rs5752634 rs5932370 rs2814536 rs2136715 rs1309824 rs9365647 rs3735912 rs7124824 rs1249772 rs12451200 rs427736 rs5932394 rs2644441 rs4855469 rs10940096 rs9355453 rs13272532 rs903209 rs1334825 rs8067882 rs5762430 rs6637420 rs1874400 rs6549411 rs2937715 rs9355454 rs17700155 rs2283257 rs1112466 rs1555142 rs470105 rs6529359 rs3011577 rs9310222 rs10064609 rs916368 rs9969608 rs2270024 rs2325364 rs8073784 rs5762461 rs5932624 rs4443876 rs2322165 rs13164793 rs6915442 rs4602925 rs4569005 rs9600257 rs2075053 rs5750726 rs1324149 rs4658702 rs2322162 rs33003 rs221725 rs7823271 rs869377 rs9573480 rs11079868 rs4821862 rs5975531 rs12407427 rs11718674 rs6874731 rs221723 rs6983626 rs11030196 rs9565288 rs747039 rs2014881 rs5954074 rs1039010 rs11921763 rs4703545 rs7789861 rs1550856 rs4923526 rs4635230 rs17637472 rs2076101 rs12687312 rs384526 rs12714781 rs2656984 rs12540575 rs3019885 rs924714 rs9517921 rs11869714 rs5758991 rs644210 rs6542651 rs6765942 rs1505000 rs4719266 rs10464861 rs1216155 rs11069386 rs739924 rs9615362 rs5919819 rs2872977 rs1319094 rs448840 rs6460807 rs7816758 rs10897108 rs9805437 rs11651374 rs9627450 rs5965846 rs934615 rs7616034 rs7733888 rs6970647 rs4593504 rs4930431 rs12428930 rs9892374 rs4459004 rs1337635 rs2110965 rs11718781 rs1449227 rs136 rs12674562 rs7118125 rs9521350 rs9907787 rs1026162 rs596987 rs7562836 rs10049211 rs4869255 rs142 rs1768869 rs7946537 rs12886280 rs11653824 rs4641225 rs580628 rs13427136 rs11128275 rs1974789 rs135 rs10511649 rs567236 rs11156803 rs17462688 rs7064826 rs995895 rs7601672 rs6808996 rs10062935 rs7783383 rs7045369 rs487345 rs12884688 rs11870700 rs1266320 rs1547727 rs10490207 rs9869897 rs10478040 rs4722417 rs10757394 rs12807746 rs1058010 rs11655981 rs6641094 rs6877 rs9309192 rs4680967 rs256249 rs6966737 rs10966093 rs7481199 rs1952198 rs7220430 rs1731475 rs12008689 rs10184594 rs1561162 rs329312 rs38488 rs824230 rs621310 rs11625855 rs7230740 rs1731470 rs1882713 rs11885364 rs729942 rs329317 rs10272887 rs928484 rs11021014 rs11159087 rs1402630 rs5955621 rs5925038 rs13427078 rs959132 rs2069882 rs739981 rs1115553 rs11021018 rs10483899 rs1940615 rs5909069 rs17253949 rs6545738 rs635268 rs17169344 rs7790537 rs3780169 rs1791459 rs1112573 rs12185468 rs6633148 rs5970118 rs1503236 rs6439419 rs2347577 rs10250368 rs17520103 rs1625749 rs12432856 rs9947725 rs2283712 rs5924705 rs6704741 rs1466827 rs10040966 rs12701041 rs495259 rs11216930 rs8008554 rs930926 rs5955648 rs5925082 rs1517862 rs2052832 rs152523 rs2392151 rs605683 rs17748 rs17259779 rs8083849 rs2122 rs4828733 rs11887934 rs347965 rs6881655 rs2598105 rs662975 rs11216943 rs1286496 rs12961249 rs6633552 rs2071122 rs4549098 rs10513320 rs1025489 rs12701773 rs567490 rs4936507 rs12893100 rs206416 rs16982185 rs3747309 rs7558702 rs391398 rs302401 rs2119053 rs985654 rs7130937 rs17099646 rs3816822 rs6653666 rs5940453 rs1459166 rs6793037 rs10516032 rs4720500 rs11142769 rs4282990 rs4906007 rs11083004 rs5925647 MitoG12373A

TABLE 16 SNP Panel of 1000 SNPs for classifying CVID case with OSAI (organ specific autoimmunity) versus CVID case without OSAI rs2297829 rs16838906 rs4274923 rs7718152 rs3912067 rs2927572 rs6589426 rs1012016 rs2850451 rs5951587 rs2275875 rs1876110 rs9291598 rs10059157 rs7805441 rs1536950 rs11214983 rs7156281 rs9949880 rs5926216 rs6699993 rs10489999 rs187196 rs7719533 rs4730345 rs957903 rs7116557 rs1840790 rs11150900 rs12011998 rs11580688 rs7562525 rs4351004 rs2400176 rs2371366 rs784790 rs7942551 rs1379230 rs11661755 rs6653559 rs1005301 rs1431900 rs10025742 rs17640419 rs17156818 rs10759209 rs4936297 rs12885991 rs12605082 rs5985835 rs12023073 rs1406665 rs10939265 rs2277025 rs2106277 rs7031681 rs7112940 rs1587351 rs12461534 rs3997122 rs533808 rs1147148 rs2687968 rs412805 rs2213979 rs10978723 rs2298767 rs4774966 rs1428752 rs5985846 rs2997447 rs1226906 rs2249563 rs394378 rs6972034 rs7871882 rs743632 rs11632793 rs10413521 rs1921501 rs3008418 rs1147155 rs2732063 rs10516028 rs17153071 rs784683 rs4937230 rs7179813 rs3786521 rs5985847 rs12069719 rs438027 rs1706023 rs868103 rs11772805 rs784676 rs7933438 rs11637130 rs6509637 rs1500727 rs2783711 rs4476347 rs1812922 rs4640812 rs1404228 rs784667 rs7948482 rs11637277 rs1366257 rs7065783 rs17257107 rs10203019 rs10518221 rs10038486 rs7777183 rs7034018 rs10790952 rs2917821 rs2561008 rs5971516 rs1325281 rs1010491 rs6857026 rs248230 rs7797250 rs7034341 rs11221388 rs12915827 rs7245993 rs2692983 rs12136144 rs6707140 rs4271959 rs9393002 rs10488631 rs13287637 rs7115037 rs8025474 rs11881864 rs2692992 rs297802 rs1446928 rs11727192 rs9379231 rs12531711 rs10306141 rs4937653 rs11071986 rs1935386 rs6527180 rs1542885 rs10173407 rs11099542 rs6934943 rs1426499 rs10987414 rs11222975 rs7172828 rs6052956 rs5927097 rs1888696 rs6708774 rs12650438 rs1905214 rs12707357 rs7047329 rs1868534 rs6495460 rs1629176 rs2748312 rs6426015 rs7422573 rs955999 rs9504663 rs918892 rs4564007 rs3016390 rs3935740 rs6085968 rs6628742 rs11264024 rs1479545 rs11731687 rs201033 rs1731913 rs4363310 rs3019650 rs1602537 rs6055504 rs16998378 rs1148945 rs2728075 rs260899 rs9383054 rs7790696 rs9411334 rs11836987 rs10220733 rs4816043 rs199905 rs784600 rs1973683 rs9307215 rs2299054 rs3807570 rs7026534 rs11047560 rs2008262 rs6133544 rs5917660 rs11211091 rs7648792 rs1215049 rs2299048 rs4725704 rs9411438 rs11171662 rs10438428 rs6055520 rs206033 rs3219484 rs359030 rs783925 rs2299045 rs903900 rs1889155 rs11171786 rs2241645 rs2179984 rs12556250 rs17265796 rs3846121 rs783963 rs3778652 rs7805359 rs7904615 rs11171949 rs12595786 rs2269027 rs5963167 rs787499 rs4685240 rs2555390 rs4510659 rs12674335 rs4880904 rs12819124 rs8037423 rs6075458 rs5963767 rs787503 rs7639807 rs2903153 rs13211187 rs12677675 rs11251234 rs1476607 rs7179643 rs6081476 rs5963172 rs12069919 rs1513755 rs6854802 rs2275906 rs12546671 rs661891 rs10875753 rs2879828 rs6046482 rs2939581 rs787497 rs4973727 rs10006414 rs16867901 rs10503603 rs10904616 rs10877323 rs7164787 rs2328472 rs2948491 rs787492 rs17007866 rs17216080 rs16867911 rs7386698 rs10904623 rs7959640 rs1828481 rs7268341 rs3008962 rs787486 rs12491696 rs9307239 rs9295739 rs1047406 rs1417032 rs1480180 rs7172789 rs7261602 rs2939564 rs12039098 rs3890082 rs9991722 rs9468202 rs1047398 rs12761228 rs1480181 rs1353576 rs6119437 rs7060889 rs1486087 rs535689 rs916913 rs6915101 rs17404749 rs12357990 rs17112001 rs11638248 rs6088393 rs12837663 rs6593584 rs681025 rs4441771 rs200959 rs1662248 rs942531 rs6539695 rs2060060 rs2038123 rs851223 rs12733869 rs680930 rs582448 rs2499450 rs327235 rs2478112 rs10466960 rs10220773 rs17332951 rs4824747 rs841669 rs549968 rs1486973 rs3814071 rs327232 rs4748050 rs7302697 rs4583204 rs10485505 rs401591 rs6689027 rs512541 rs4293833 rs3777543 rs1481758 rs2025718 rs1543169 rs1431234 rs760998 rs241748 rs9428050 rs537660 rs586090 rs1507912 rs1545961 rs9651540 rs2056218 rs4313781 rs6072090 rs241742 rs553314 rs9310709 rs7696023 rs3957360 rs16879142 rs11012422 rs7960163 rs6496332 rs11086875 rs241740 rs10735234 rs9826441 rs12504357 rs239794 rs13282541 rs11598403 rs10778309 rs10520630 rs12479650 rs17302689 rs499345 rs9818043 rs3796590 rs9345994 rs11994501 rs11009254 rs7976552 rs715922 rs911952 rs5951221 rs21327 rs7627134 rs7668220 rs6455228 rs1568135 rs6481837 rs11068326 rs2163442 rs666753 rs4986620 rs1814653 rs2372250 rs10030326 rs1157977 rs1517118 rs12768519 rs10850758 rs2448929 rs6513239 rs5951181 rs2769267 rs7612515 rs4691394 rs1025534 rs1549459 rs4240498 rs10850759 rs731597 rs1007725 rs4986597 rs12410158 rs6772285 rs6840827 rs1655365 rs7014791 rs16937609 rs10850760 rs1371390 rs4812180 rs5951214 rs1027493 rs11918364 rs7694824 rs13205488 rs7004470 rs7918437 rs7299612 rs1025768 rs1201916 rs4986610 rs6672415 rs4955377 rs17534473 rs1890278 rs7815631 rs6481383 rs7314278 rs1371391 rs13042560 rs7055300 rs6427832 rs7637194 rs17475331 rs9373923 rs7815950 rs1867571 rs1268892 rs10048024 rs2426977 rs2428414 rs3738287 rs9853319 rs6825169 rs9487538 rs3016889 rs1416764 rs838918 rs11635251 rs1884854 rs239902 rs10800777 rs11922288 rs4695771 rs17679624 rs2470002 rs714113 rs838916 rs8039888 rs2426987 rs6616403 rs10800779 rs11713329 rs7659511 rs17057836 rs2470012 rs1416767 rs826723 rs757108 rs6015934 rs5922869 rs823105 rs6599210 rs1347700 rs17185166 rs2447167 rs3851235 rs10848159 rs2283479 rs6129079 rs825541 rs3890801 rs4676595 rs4362867 rs4897579 rs11778912 rs10999626 rs10751692 rs233948 rs6028076 rs1531868 rs11587049 rs9868689 rs869570 rs1209415 rs4242335 rs11001790 rs12319878 rs7186783 rs1735484 rs5968255 rs7546886 rs2191028 rs6883247 rs726794 rs7813708 rs1907333 rs2476509 rs12927837 rs2822744 rs10218349 rs11590870 rs2191027 rs1875195 rs11965233 rs11779458 rs1907303 rs520000 rs933488 rs7282227 rs765076 rs7542307 rs4299518 rs4075798 rs9376674 rs4512366 rs17772401 rs348222 rs2312949 rs2824332 rs7878135 rs753961 rs6794124 rs10512785 rs4895598 rs3812475 rs3847452 rs9532874 rs4781120 rs2824335 rs5968299 rs7367845 rs724334 rs493944 rs6930478 rs4128468 rs10509532 rs9532877 rs7403964 rs17801179 rs5968301 rs4304564 rs1911746 rs12153548 rs13191080 rs2648836 rs10788575 rs1555587 rs8048521 rs2833979 rs6418331 rs1079344 rs1406611 rs31887 rs4870010 rs2648847 rs7084813 rs9535207 rs7199993 rs9974198 rs1000505 rs10489802 rs9878560 rs7710236 rs7745737 rs2909239 rs6583692 rs9536203 rs16960659 rs2252046 rs5939564 rs12353954 rs4131544 rs7706312 rs851991 rs2975520 rs7911956 rs9569174 rs2519975 rs9979659 rs7050797 rs4649442 rs3910485 rs27466 rs890458 rs11166726 rs2147878 rs2316640 rs8055835 rs2849684 rs675270 rs1881548 rs9831003 rs32376 rs645851 rs7845645 rs12766594 rs4274330 rs3213881 rs9306194 rs595679 rs6678625 rs4855779 rs10066628 rs9347586 rs979289 rs2487999 rs2321995 rs7191446 rs5747145 rs10521528 rs17640830 rs1437238 rs1494621 rs12197625 rs10107665 rs723483 rs4132047 rs11648791 rs5746398 rs12855355 rs887962 rs12638978 rs4301212 rs1476041 rs6578102 rs12415142 rs17080881 rs9939233 rs1004973 rs676579 rs2380431 rs6774951 rs2662266 rs903791 rs10759031 rs1538080 rs10507731 rs8050239 rs5747176 rs640009 rs6733870 rs17826288 rs7708845 rs9365899 rs324526 rs7919329 rs1023033 rs4405545 rs4819591 rs11152747 rs7564116 rs709496 rs1500238 rs6455419 rs88818 rs10128498 rs17082395 rs7202571 rs5992761 rs6643108 rs6744117 rs709477 rs6594343 rs1626670 rs12156538 rs9418990 rs1537547 rs8063008 rs134510 rs12839137 rs4669995 rs697957 rs17412225 rs10230651 rs649891 rs2141322 rs9565348 rs1476460 rs9609551 rs5905284 rs11895101 rs10511323 rs1423456 rs12670364 rs10756313 rs217766 rs7320056 rs17806045 rs670264 rs4825645 rs4669999 rs4682479 rs7711677 rs7797010 rs1978746 rs7936055 rs2775133 rs205043 rs728797 rs6603535 rs1395 rs1837206 rs7714521 rs1505335 rs4401960 rs17761158 rs1324770 rs1870584 rs5915542 rs5911118 rs10496179 rs6806321 rs42670 rs12703012 rs7037514 rs10838249 rs2026840 rs4796033 rs5916081 rs16996610 rs13021944 rs4234209 rs6871668 rs2107140 rs10811312 rs7939723 rs1324791 rs2280784 rs11094994 rs2840649 rs2177098 rs1384566 rs13354840 rs17164591 rs7866455 rs10791889 rs6563154 rs12103666 rs7055627 rs983007 rs2140039 rs4679142 rs1487216 rs11771731 rs7854529 rs4930390 rs1113104 rs9899005 rs1450154 rs12557782 rs2861680 rs970494 rs10056192 rs1019171 rs1563950 rs3741194 rs4884202 rs4792901 rs1450152 rs5932693 rs10520218 rs6807545 rs7726250 rs10950426 rs13290599 rs7104011 rs1535989 rs11655764 rs5989530 rs4269687 rs1992886 rs12487078 rs10077150 rs1548741 rs13297389 rs12421802 rs9582985 rs12953076 rs5989531 rs2886973 rs6748174 rs13093879 rs9765600 rs4721160 rs1013217 rs1151220 rs9520140 rs792766 rs1026162 rs4829963 rs3218984 rs9864846 rs1524566 rs217601 rs2481061 rs2373711 rs9555701 rs2049534 rs1015496 rs5974805 rs7589525 rs524554 rs6865072 rs36843 rs10813971 rs10897739 rs17122962 rs7210515 rs5978969 rs5908057 rs13401717 rs531617 rs13167682 rs36920 rs13287406 rs7127610 rs1950950 rs9892439 rs5978974 rs1120947 rs11685896 rs7625382 rs2218717 rs10227084 rs616009 rs7948940 rs1570164 rs7218480 rs2404797 rs2223457 rs1442774 rs9827666 rs1524560 rs1476944 rs7861989 rs3824933 rs10483412 rs10512540 rs6530209 rs11798497 rs4849721 rs9833887 rs1524559 rs2192484 rs776021 rs4396256 rs8012446 rs898095 rs17222040 rs5908792 rs1527420 rs7618020 rs1524561 rs9648259 rs4878743 rs11224780 rs12586232 rs7209936 rs929228 rs5953883 rs4662982 rs9879759 rs6891778 rs985307 rs1421156 rs7948300 rs1571106 rs16941996 rs6638877 rs6654220 rs2677646 rs2242338 rs716815 rs4721784 rs656875 rs11822192 rs6573233 rs6506135 rs5935341 rs5908801 rs12475202 rs12495729 rs1992951 rs6461547 rs1899937 rs11224783 rs10139749 rs8083753 rs5935343 rs5936357 rs10930958 rs6799976 rs10477215 rs1859103 rs7863238 rs12362157 rs741518 rs675881 rs3863537 rs4090449 rs1962276 rs13086529 rs13156143 rs9638797 rs11143520 rs7117447 rs2619676 rs676968 rs11095604 rs12388487 rs12474703 rs16859685 rs325231 rs12700427 rs2479835 rs7124340 rs162171 rs1403753 rs5934075 rs5936468 rs6754105 rs12493115 rs17415614 rs882101 rs12351660 rs4754161 rs327434 rs3861809 rs1921931 rs237370 rs5008216 rs4687488 rs2636112 rs2074137 rs7855727 rs17105869 rs162174 rs17064241 rs6527755 rs3761562 rs1037091 rs7637593 rs1096980 rs7779755 rs10867947 rs4245155 rs7158936 rs4362477 rs5909379 rs1472977 rs10185386 rs4527429 rs3792815 rs6978786 rs10115014 rs11601037 rs1617580 rs7231765 rs12850925 rs4898398

Example 3 Exome Sequencing Identifies Missense IRF2BP2 Mutation in a Family with Autosomal Dominant Common Variable Immunodeficiency (CVID)

Common Variable Immunodeficiency (CVID) is among the most frequently diagnosed forms of primary immunodeficiency disorders (PIDD), and is the most frequent requiring clinical intervention. Defined clinically by low quantity of two immunoglobulin classes (including IgG) and poor specific antibody production, it has been thought of as an “umbrella diagnosis” due to the heterogeneity of its onset and co-morbidities, including autoimmune disease and risk of malignancy. Though thought to be polygenic, roughly 10% of CVID is familial, and in recent years, 11 gene causes and/or associations with CVID have been described (Table 17).

TABLE 17 CVID Gene Associations Function Gene Ref. BCR function CD19 Van Zelm, MC, et al. NEJM 2006 CD20 Kuijpers TW, et al. JCI 2010 CD21 Thiel J, et al. JACI 2012 CD81 Van Zelm MC, et al. JCI 2010. B-cell TACI Martinez-Gallo M, et al. JACI 2013 co-stimulation ICOS Grimbacher B, et al. Nat Immunol 2003 BAFFR Warnatz H, et al. Proc Natl Acad Sci 2009 CD27 Van Montfrans J, et al. JACI 2012 DNA repair/ MSH5 Sekine H, et al. Proc Natl Acad Sci 2007 VDJ recombination B-cell survival/ LRBA Lopez-Herrera G, et al. Am J Hum Genet 2012 differentiation TCR/BCR PLCγ2 Ombrello MJ, et al. NEJM 2012 signaling

As discussed above in the previous examples, a recent multi-institutional genome-wide array study of CVID showed unique associations with specific single nucleotide polymorphisms (SNP) and copy number variants (CNV), with intraexonic duplications in ORC4L being most highly associated with disease. Beyond individual associations, CVID has a unique pattern of SNP and CNV, as suggested by the successful use of a Support Vector Machine (SVM) algorithm to identify this pattern in CVID patients and controls. SVM can be trained with a variety of data, and produces a “hyperplane” for subsequent classification. In the studies described above, the CVID SVM hyperplane successfully classified cases with an accuracy of 91%, PPV 100%, and NPV 96%.

Though the use of SVM supports the polygenic nature of CVID, a remaining question was whether monogenic CVID would lack the genetic fingerprint of the more common polygenic disease. If this were the case, SVM classification of microarray data would incorrectly classify these individuals as not affected, and could therefore be a useful screening method for monogenic CVID, most notably in familial cases. Accordingly additional studies were undertaken.

Patients

Patients were enrolled in research protocols approved by the host institutional review board to allow genetic analysis and clinical data collection.

Clinical History

The proband is a young woman who experienced recurrent sinopulmonary infections beginning in early adolescence. Evaluation at age 19 years showed IgG 620 mg/dl (normal range 635-1775 mg/dL), undetectable IgM and IgA (normal ranges 71-237 and 70-486 mg/dL respectively), and poor specific antibody production following vaccination to S. pneumoniae (1 of 14 serotypes >1.3 mcg/ml) and N. meningitidis (0 of 4 strains protective). Lymphocyte subsets were normal in number with CD19⁺ 388 cell/mcl (normal range 100-570), though switched memory B-cells (CD19⁺IgD⁻CD27⁺) were low at 0.4% (normal >0.5%). She has been maintained on subcutaneous immunoglobulin with good control of infections, and has no autoimmune disease.

Her family history is notable for CVID in both her father as well as her older brother (FIG. 11). Her father also has a history of psoriasis, and her brother has type I diabetes. Her paternal grandparents, aunts, and their families are unaffected, as is her maternal family.

Genetic Studies

High throughput SNP genotyping was performed with the Infinitium HumanHap610 Beadchip, and the PennCNV algorithm was used for CNV calls. A support vector machine algorithm was trained with data from 179 CVID cases and 1917 controls, utilizing the 658 most significantly associated variants from the 2011 study, and subsequently tested on the patient.

Whole-exome sequencing using the Agilent SureSelect Human All Exon 50 Mb kit was performed on the patient and her family. Variants were matched to disease segregation (which suggested a heterozygous, autosomal dominant pattern), and further narrowed by exclusion of synonymous mutations, in silico analysis of mutation impact, exclusion of SNPs in public databases (1000 Genomes and NHLBI 5400 exomes Project), tissue expression pattern (BioGPS.org), and ties to known immunologic pathways.

RT-PCR and Immunoblotting

RNA was isolated from whole blood obtained from the patient and controls via Trizol reagent (Applied Biosystems, Grand Island, N.Y.) and RNEasy kit (Qiagen, Germantown, Md.). cDNA was produced via high capacity Reverse Transcriptase kit (Applied Biosystems), and custom cDNA primers for IRF2BP2 (both total and isoform 2) and GAPDH were created. RT-PCR performed via SYBR Green core reagents on a QPCR 7900HT system. Gene dose was calculated via DDCT method.

Protein immunoblotting was performed on lymphoblasts derived from peripheral blood mononuclear cells via treatment with phytohemagglutanin (2 ug/ml) and IL-2 (500 IU/ml). Antibodies against IRF2BP2 and TATA binding protein (Abcam, Cambridge, Mass.) were used.

Support Vector Machine Hyperplane Classification

Analysis of the top 658 SNP/CNVs by the pre-trained CVID SVM algorithm predicted that the patient was not affected (FIG. 11).

Whole Exome Sequencing

Sequencing of the proband revealed 12 non-synonymous, rare variants, of which 5 were predicted to be damaging via in silico analysis (SIFT, polyphen2). Three variants (AMBP, IRF2BP2, and PIK3C2G) had been tied to immunologic pathways. Sanger sequencing of IRF2BP2 in the proband and family confirmed that a heterozygous S535N mutation was present in all affected family members, and none of those who were unaffected. This mutation is in the RING domain of the protein, which has been described as the region of interaction with IRF2 and NFAT1 [3]. See FIG. 12.

RT-PCR of IRF2BP2

Quantitative PCR of IRF2BP2 from patient and control peripheral blood showed a lower transcript quantity in the patient versus normal control (FIG. 13). In the normal control, Isoform 1 was predominant, with comparatively less transcripts of Isoform 2 (which differs by exon 1 length). In the patient, isoform expression was roughly equal.

These results reveal that learning algorithms such as SVM provide a valuable method of screening for monogenic diseases within polygenic populations by searching for differences in the genetic pattern of SNP and rare CNV. In the field of PIDD, this is particularly useful for screening for monogenic causes of early-onset autoimmune disease such as type I diabetes or inflammatory bowel disease.

Moreover, these studies reveal that the presence of a SNP in IRF2BP2 on chromosome 1 at position 234742995 is indicative of a new CVID-associated candidate gene.

Example 4

The information herein above can be applied clinically to patients for diagnosing an increased susceptibility for developing CVID, and therapeutic intervention. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. Diagnostic compositions, including microarrays, and methods can be designed to identify the genetic alterations described herein in nucleic acids from a patient to assess susceptibility for developing CVID. This can occur after a patient arrives in the clinic; the patient has blood drawn, and using the diagnostic methods described herein, a clinician can detect one or more indicative SNPs or CNVs described in the Tables hereinabove. The information obtained from the patient sample, which can optionally be amplified prior to assessment, will be used to diagnose a patient with an increased or decreased susceptibility for developing CVID. Kits for performing the diagnostic method of the invention are also provided herein. Such kits comprise a microarray comprising at least one of the SNPs provided herein in and the necessary reagents for assessing the patient samples as described above.

The identity of CVID-involved genes and the patient results will indicate which variants are present, and will identify those that possess an altered risk for developing CVID. The information provided herein allows for therapeutic intervention at earlier times in disease progression that previously possible. For example, agents such as STA-5326, sotatercept, PF3446962, PEG-interleukin-2 and B-lymphocyte stimulators can be administered as appropriate.

Also as described herein above, several new genes involved in CVID pathogenesis have been identified which provide novel targets for the development of new therapeutic agents efficacious for the treatment of CVID.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope of the present invention, as set forth in the following claims. 

What is claimed is:
 1. A solid support for detecting at least one common variable immunodeficiency (CVID)-associated copy number variation (CNV) in a sample comprising immobilized nucleic acid probes or primers of 15 or more nucleotides in length, wherein each of said probes or primers present on said solid support specifically hybridize to CVID specific nucleic acids in chromosomal regions consisting of chr11: 85365857-85381622, chr20: 57735790-57741780, chr22: 17396663-18417315, chr4: 10256682-10264316, and chr10: 46003146-46042543, chr2: 148396730-148433180, chr15: 35053039-35063531, chr7: 91789778-91801963, chr2: 163316298-163316595, chr7: 87180702-87191931, and, optionally, one or more of chr19: 9256584-9277749, chr4: 39190766-39201960, chr7: 18903915-18905725, chr5: 170531269-170536993, chr7: 34685030-34686172, and chr1: 170400944-170403742.
 2. A kit comprising the solid support of claim 1 and reagents for detecting hybridization of nucleic acids to said solid support. 